Novel crispr enzymes and systems

ABSTRACT

The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered RNA-targeting systems comprising a novel RNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication Nos. 62/320,231, filed Apr. 8, 2016, 62/181,675, filed Jun.18, 2015, 62/285,349, filed Oct. 22, 2015, 62/296,522, filed Feb. 17,2016, and 62/320,231, filed Apr. 8, 2016.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbersMH100706, MH110049, DK097768 and GM10407 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

All documents cited or referenced in herein cited documents, togetherwith any manufacturer's instructions, descriptions, productspecifications, and product sheets for any products mentioned herein orin any document incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention. More specifically, all referenced documents areincorporated by reference to the same extent as if each individualdocument was specifically and individually indicated to be incorporatedby reference.

FIELD OF THE INVENTION

The present invention generally relates to systems, methods andcompositions used for the control of gene expression involving sequencetargeting, such as perturbation of gene transcripts or nucleic acidediting, that may use vector systems related to Clustered RegularlyInterspaced Short Palindromic Repeats (CRISPR) and components thereof.

BACKGROUND OF THE INVENTION

Recent advances in genome sequencing techniques and analysis methodshave significantly accelerated the ability to catalog and map geneticfactors associated with a diverse range of biological functions anddiseases. Precise genome targeting technologies are needed to enablesystematic reverse engineering of causal genetic variations by allowingselective perturbation of individual genetic elements, as well as toadvance synthetic biology, biotechnological, and medical applications.Although genome-editing techniques such as designer zinc fingers,transcription activator-like effectors (TALEs), or homing meganucleasesare available for producing targeted genome perturbations, there remainsa need for new genome and transcriptome engineering technologies thatemploy novel strategies and molecular mechanisms and are affordable,easy to set up, scalable, and amenable to targeting multiple positionswithin the eukaryotic genome and transcriptome. This would provide amajor resource for new applications in genome engineering andbiotechnology.

The CRISPR-Cas systems of bacterial and archaeal adaptive immunity showextreme diversity of protein composition and genomic loci architecture.The CRISPR-Cas system loci has more than 50 gene families and there isno strictly universal genes indicating fast evolution and extremediversity of loci architecture. So far, adopting a multi-prongedapproach, there is comprehensive cas gene identification of about 395profiles for 93 Cas proteins. Classification includes signature geneprofiles plus signatures of locus architecture. A new classification ofCRISPR-Cas systems is proposed in which these systems are broadlydivided into two classes, Class 1 with multisubunit effector complexesand Class 2 with single-subunit effector modules exemplified by the Cas9protein (FIGS. 1A and 1B). Novel effector proteins associated with Class2 CRISPR-Cas systems may be developed as powerful genome engineeringtools and the prediction of putative novel effector proteins and theirengineering and optimization is important.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

The CRISPR-Cas adaptive immune system defends microbes against foreigngenetic elements via DNA or RNA-DNA interference. Here, we interrogatethe Class 2 type VI single-component CRISPR-Cas effector C2c2 andcharacterize it as an RNA-guided RNase. We demonstrate that C2c2 (e.g.from Leptotrichia shahii) provides robust interference against RNA phageinfection. Through in vitro biochemical analysis and in vivo assays, weshow that C2c2 can be programmed to cleave ssRNA targets carryingprotospacers flanked by a 3′ H (non-G) PAM. Cleavage is mediated bycatalytic residues in the two conserved HEPN domains of C2c2, mutationsin which generate a catalytically inactive RNA-binding protein. C2c2 isguided by a single crRNA and can be re-programmed to deplete specificmRNAs in vivo. We show that LshC2c2 can be targeted to a specific siteof interest and can carry out non-specific RNase activity once primedwith the cognate target RNA. These results broaden our understanding ofCRISPR-Cas systems and demonstrate the possibility of harnessing C2c2 todevelop a broad set of RNA-targeting tools.

There exists a pressing need for alternative and robust systems andtechniques for targeting nucleic acids or polynucleotides (e.g. DNA orRNA or any hybrid or derivative thereof) with a wide array ofapplications. This invention addresses this need and provides relatedadvantages. Adding the novel DNA or RNA-targeting systems of the presentapplication to the repertoire of genomic and epigenomic targetingtechnologies may transform the study and perturbation or editing ofspecific target sites through direct detection, analysis andmanipulation. To utilize the DNA or RNA-targeting systems of the presentapplication effectively for genomic or epigenomic targeting withoutdeleterious effects, it is critical to understand aspects of engineeringand optimization of these DNA or RNA targeting tools.

The Class 2 type VI effector protein C2c2 is a RNA-guided RNase that canbe efficiently programmed to degrade ssRNA. C2c2 achieves RNA cleavagethrough conserved basic residues within its two HEPN domains, incontrast to the catalytic mechanisms of other known RNases found inCRISPR-Cas systems. Mutation of the HEPN domain, such as (e.g. alanine)substitution, of any of the four predicted HEPN domain catalyticresidues converted C2c2 into an inactive programmable RNA-bindingprotein (dC2c2, analogous to dCas9).

The ability of dC2c2 to bind to specified sequences could be used inseveral aspects according to the invention to (i) bring effector modulesto specific transcripts to modulate the function or translation, whichcould be used for large-scale screening, construction of syntheticregulatory circuits and other purposes; (ii) fluorescently tag specificRNAs to visualize their trafficking and/or localization; (iii) alter RNAlocalization through domains with affinity for specific subcellularcompartments; and (iv) capture specific transcripts (through direct pulldown of dC2c2 or use of dC2c2 to localize biotin ligase activity tospecific transcripts) to enrich for proximal molecular partners,including RNAs and proteins.

Active C2c2 should also have many applications. An aspect of theinvention involves targeting a specific transcript for destruction, aswith RFP here. In addition, C2c2, once primed by the cognate target, cancleave other (non-complementary) RNA molecules in vitro and can inhibitcell growth in vivo. Biologically, this promiscuous RNase activity mayreflect a programmed cell death/dormancy (PCD/D)-based protectionmechanism of the type VI CRISPR-Cas systems. Accordingly, in an aspectof the invention, it might be used to trigger PCD or dormancy inspecific cells—for example, cancer cells expressing a particulartranscript, neurons of a given class, cells infected by a specificpathogen, or other aberrant cells or cells the presence of which isotherwise undesirable.

The invention provides a method of modifying nucleic acid sequencesassociated with or at a target locus of interest, the method comprisingdelivering to said locus a non-naturally occurring or engineeredcomposition comprising a Type VI CRISPR-Cas loci effector protein andone or more nucleic acid components, wherein the effector protein formsa complex with the one or more nucleic acid components and upon bindingof the said complex to the locus of interest the effector proteininduces the modification of the sequences associated with or at thetarget locus of interest. In a preferred embodiment, the modification isthe introduction of a strand break. In a preferred embodiment, thesequences associated with or at the target locus of interest comprisesRNA or DNA and the effector protein is encoded by a type VI CRISPR-Casloci.

It will be appreciated that the terms Cas enzyme, CRISPR enzyme, CRISPRprotein, Cas protein and CRISPR Cas are generally used interchangeablyand at all points of reference herein refer by analogy to novel CRISPReffector proteins further described in this application, unlessotherwise apparent, such as by specific reference to Cas9. The CRISPReffector proteins described herein are preferably C2c2 effectorproteins.

The invention provides a method of modifying sequences associated withor at a target locus of interest, the method comprising delivering tosaid sequences associated with or at the locus a non-naturally occurringor engineered composition comprising a C2c2 loci effector protein andone or more nucleic acid components, wherein the C2c2 effector proteinforms a complex with the one or more nucleic acid components and uponbinding of the said complex to the locus of interest the effectorprotein induces the modification of sequences associated with or at thetarget locus of interest. In a preferred embodiment, the modification isthe introduction of a strand break. In a preferred embodiment the C2c2effector protein forms a complex with one nucleic acid component;advantageously an engineered or non-naturally occurring nucleic acidcomponent. The induction of modification of sequences associated with orat the target locus of interest can be C2c2 effector protein-nucleicacid guided. In a preferred embodiment the one nucleic acid component isa CRISPR RNA (crRNA). In a preferred embodiment the one nucleic acidcomponent is a mature crRNA or guide RNA, wherein the mature crRNA orguide RNA comprises a spacer sequence (or guide sequence) and a directrepeat sequence or derivatives thereof. In a preferred embodiment thespacer sequence or the derivative thereof comprises a seed sequence,wherein the seed sequence is critical for recognition and/orhybridization to the sequence at the target locus. In a preferredembodiment, the sequences associated with or at the target locus ofinterest comprise linear or super coiled DNA.

Aspects of the invention relate to C2c2 effector protein complexeshaving one or more non-naturally occurring or engineered or modified oroptimized nucleic acid components. In a preferred embodiment the nucleicacid component of the complex may comprise a guide sequence linked to adirect repeat sequence, wherein the direct repeat sequence comprises oneor more stem loops or optimized secondary structures. In certainembodiments, the direct repeat has a minimum length of 16 nts, such asat least 28 nt, and a single stem loop. In further embodiments thedirect repeat has a length longer than 16 nts, preferably more than 17nts, such as at least 28 nt, and has more than one stem loop oroptimized secondary structures. In particular embodiments, the directrepeat has 25 or more nts, such as 26 nt, 27 nt, 28 nt or more, and oneor more stem loop structures. In a preferred embodiment the directrepeat may be modified to comprise one or more protein-binding RNAaptamers. In a preferred embodiment, one or more aptamers may beincluded such as part of optimized secondary structure. Such aptamersmay be capable of binding a bacteriophage coat protein. Thebacteriophage coat protein may be selected from the group comprising Qβ,F2, GA, fr, JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1,TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r,7s and PRR1. In a preferred embodiment the bacteriophage coat protein isMS2. The invention also provides for the nucleic acid component of thecomplex being 30 or more, 40 or more or 50 or more nucleotides inlength.

The invention provides methods of genome editing and transcriptomeperturbation wherein the method comprises two or more rounds of C2c2effector protein targeting and cleavage. In certain embodiments, a firstround comprises the C2c2 effector protein cleaving sequences associatedwith a target locus far away from the seed sequence and a second roundcomprises the C2c2 effector protein cleaving sequences at the targetlocus. In certain such embodiments of the invention, a first round oftargeting by a C2c2 effector protein results in a strand break and asecond round of targeting by the C2c2 effector protein results in asecond strand break. In an embodiment of the invention, one or morerounds of targeting by a C2c2 effector protein results in staggeredcleavage that may be repaired.

The invention also provides a method of modifying a target locus ofinterest, the method comprising delivering to said locus a non-naturallyoccurring or engineered composition comprising a C2c2 loci effectorprotein and one or more nucleic acid components, wherein the C2c2effector protein forms a complex with the one or more nucleic acidcomponents and upon binding of the said complex to the locus of interestthe effector protein induces the modification of the target locus ofinterest. In a preferred embodiment, the modification is theintroduction of a strand break.

In such methods the target locus of interest may be comprised within anRNA moledule. Also, the target locus of interest may be comprised withina DNA molecule, and in certain embodiments, within a transcribed DNAmolecule. In such methods the target locus of interest may be comprisedin a nucleic acid molecule in vitro.

In such methods the target locus of interest may be comprised in anucleic acid molecule within a cell. The cell may be a prokaryotic cellor a eukaryotic cell. The cell may be a mammalian cell. The mammaliancell many be a non-human primate, bovine, porcine, rodent or mouse cell.The cell may be a non-mammalian eukaryotic cell such as poultry, fish orshrimp. The cell may also be a plant cell. The plant cell may be of acrop plant such as cassava, corn, sorghum, wheat, or rice. The plantcell may also be of an algae, tree or vegetable. The modificationintroduced to the cell by the present invention may be such that thecell and progeny of the cell are altered for improved production ofbiologic products such as an antibody, starch, alcohol or other desiredcellular output. The modification introduced to the cell by the presentinvention may be such that the cell and progeny of the cell include analteration that changes the biologic product produced.

The mammalian cell many be a non-human mammal, e.g., primate, bovine,ovine, porcine, canine, rodent, Leporidae such as monkey, cow, sheep,pig, dog, rabbit, rat or mouse cell. The cell may be a non-mammalianeukaryotic cell such as poultry bird (e.g., chicken), vertebrate fish(e.g., salmon) or shellfish (e.g., oyster, claim, lobster, shrimp) cell.The cell may also be a plant cell. The plant cell may be of a monocot ordicot or of a crop or grain plant such as cassava, corn, sorghum,soybean, wheat, oat or rice. The plant cell may also be of an algae,tree or production plant, fruit or vegetable (e.g., trees such as citrustrees, e.g., orange, grapefruit or lemon trees; peach or nectarinetrees; apple or pear trees; nut trees such as almond or walnut orpistachio trees; nightshade plants; plants of the genus Brassica; plantsof the genus Lactuca; plants of the genus Spinacia; plants of the genusCapsicum; cotton, tobacco, asparagus, carrot, cabbage, broccoli,cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry,blueberry, raspberry, blackberry, grape, coffee, cocoa, etc).

The invention provides a method of modifying a target locus of interest,the method comprising delivering to said locus a non-naturally occurringor engineered composition comprising a Type VI CRISPR-Cas loci effectorprotein and one or more nucleic acid components, wherein the effectorprotein forms a complex with the one or more nucleic acid components andupon binding of the said complex to the locus of interest the effectorprotein induces the modification of the target locus of interest. In apreferred embodiment, the modification is the introduction of a strandbreak.

In such methods the target locus of interest may be comprised within aDNA molecule or within an RNA molecule. In a preferred embodiment, thetarget locus of interest comprises RNA.

The invention also provides a method of modifying a target locus ofinterest, the method comprising delivering to said locus a non-naturallyoccurring or engineered composition comprising a C2c2 loci effectorprotein and one or more nucleic acid components, wherein the C2c2effector protein forms a complex with the one or more nucleic acidcomponents and upon binding of the said complex to the locus of interestthe effector protein induces the modification of the target locus ofinterest. In a preferred embodiment, the modification is theintroduction of a strand break.

In such methods the target locus of interest may be comprised in anucleic acid molecule in vitro. In such methods the target locus ofinterest may be comprised in a nucleic acid molecule within a cell.Preferably, in such methods the target locus of interest may becomprised in a RNA molecule in vitro. Also preferably, in such methodsthe target locus of interest may be comprised in a RNA molecule within acell. The cell may be a prokaryotic cell or a eukaryotic cell. The cellmay be a mammalian cell. The cell may be a rodent cell. The cell may bea mouse cell.

In any of the described methods the target locus of interest may be agenomic or epigenomic locus of interest. In any of the described methodsthe complex may be delivered with multiple guides for multiplexed use.In any of the described methods more than one protein(s) may be used.

In further aspects of the invention the nucleic acid components maycomprise a putative CRISPR RNA (crRNA) sequence. Without limitation, theApplicants hypothesize that in such instances, the pre-crRNA maycomprise secondary structure that is sufficient for processing to yieldthe mature crRNA as well as crRNA loading onto the effector protein. Bymeans of example and not limitation, such secondary structure maycomprise, consist essentially of or consist of a stem loop within thepre-crRNA, more particularly within the direct repeat.

In any of the described methods the effector protein and nucleic acidcomponents may be provided via one or more polynucleotide moleculesencoding the protein and/or nucleic acid component(s), and wherein theone or more polynucleotide molecules are operably configured to expressthe protein and/or the nucleic acid component(s). The one or morepolynucleotide molecules may comprise one or more regulatory elementsoperably configured to express the protein and/or the nucleic acidcomponent(s). The one or more polynucleotide molecules may be comprisedwithin one or more vectors. In any of the described methods the targetlocus of interest may be a genomic or epigenomic locus of interest. Inany of the described methods the complex may be delivered with multipleguides for multiplexed use. In any of the described methods more thanone protein(s) may be used.

In any of the described methods the strand break may be a single strandbreak or a double strand break.

Regulatory elements may comprise inducible promotors. Polynucleotidesand/or vector systems may comprise inducible systems.

In any of the described methods the one or more polynucleotide moleculesmay be comprised in a delivery system, or the one or more vectors may becomprised in a delivery system.

In any of the described methods the non-naturally occurring orengineered composition may be delivered via liposomes, particlesincluding nanoparticles, exosomes, microvesicles, a gene-gun or one ormore viral vectors.

The invention also provides a non-naturally occurring or engineeredcomposition which is a composition having the characteristics asdiscussed herein or defined in any of the herein described methods.

In certain embodiments, the invention thus provides a non-naturallyoccurring or engineered composition, such as particularly a compositioncapable of or configured to modify a target locus of interest, saidcomposition comprising a Type VI CRISPR-Cas loci effector protein andone or more nucleic acid components, wherein the effector protein formsa complex with the one or more nucleic acid components and upon bindingof the said complex to the locus of interest the effector proteininduces the modification of the target locus of interest. In certainembodiments, the effector protein may be a C2c2 loci effector protein.

The invention also provides in a further aspect a non-naturallyoccurring or engineered composition, such as particularly a compositioncapable of or configured to modify a target locus of interest, saidcomposition comprising: (a) a guide RNA molecule (or a combination ofguide RNA molecules, e.g., a first guide RNA molecule and a second guideRNA molecule) or a nucleic acid encoding the guide RNA molecule (or oneor more nucleic acids encoding the combination of guide RNA molecules);(b) a Type VI CRISPR-Cas loci effector protein or a nucleic acidencoding the Type VI CRISPR-Cas loci effector protein. In certainembodiments, the effector protein may be a C2c2 loci effector protein.

The invention also provides in a further aspect a non-naturallyoccurring or engineered composition comprising: (a) a guide RNA molecule(or a combination of guide RNA molecules, e.g., a first guide RNAmolecule and a second guide RNA molecule) or a nucleic acid encoding theguide RNA molecule (or one or more nucleic acids encoding thecombination of guide RNA molecules); (b) be a C2c2 loci effectorprotein.

The invention also provides a vector system comprising one or morevectors, the one or more vectors comprising one or more polynucleotidemolecules encoding components of a non-naturally occurring or engineeredcomposition which is a composition having the characteristics as definedin any of the herein described methods.

The invention also provides a delivery system comprising one or morevectors or one or more polynucleotide molecules, the one or more vectorsor polynucleotide molecules comprising one or more polynucleotidemolecules encoding components of a non-naturally occurring or engineeredcomposition which is a composition having the characteristics discussedherein or as defined in any of the herein described methods.

The invention also provides a non-naturally occurring or engineeredcomposition, or one or more polynucleotides encoding components of saidcomposition, or vector or delivery systems comprising one or morepolynucleotides encoding components of said composition for use in atherapeutic method of treatment. The therapeutic method of treatment maycomprise gene or transcriptome editing, or gene therapy.

The invention also encompasses computational methods and algorithms topredict new Class 2 CRISPR-Cas systems and identify the componentstherein.

The invention also provides for methods and compositions wherein one ormore amino acid residues of the effector protein may be modified e.g.,an engineered or non-naturally-occurring effector protein or C2c2. In anembodiment, the modification may comprise mutation of one or more aminoacid residues of the effector protein. The one or more mutations may bein one or more catalytically active domains of the effector protein. Theeffector protein may have reduced or abolished nuclease activitycompared with an effector protein lacking said one or more mutations.The effector protein may not direct cleavage of one or other DNA or RNAstrand at the target locus of interest. The effector protein may notdirect cleavage of either DNA or RNA strand at the target locus ofinterest. In a preferred embodiment, the one or more mutations maycomprise two mutations. In a preferred embodiment the one or more aminoacid residues are modified in a C2c2 effector protein, e.g., anengineered or non-naturally-occurring effector protein or C2c2. Inparticular embodiments, the one or more modified of mutated amino acidresidues are one or more of those in C2c2 corresponding to R597, H602,R1278 and H1283 (referenced to Lsh C2c2 amino acids and C2c2 consensusnumbering), such as mutations R597A, H602A, R1278A and H1283A, or thecorresponding amino acid residues in Lsh C2c2 orthologues.

In particular embodiments, the one or more modified of mutated aminoacid residues are one or more of those in C2c2 corresponding to K2, K39,V40, E479, L514, V518, N524, G534, K535, E580, L597, V602, D630, F676,L709, I713, R717 (HEPN), N718, H722 (HEPN), E773, P823, V828, I879,Y880, F884, Y997, L1001, F1009, L1013, Y1093, L1099, L1111, Y1114,L1203, D1222, Y1244, L1250, L1253, K1261, I1334, L1355, L1359, R1362,Y1366, E1371, R1372, D1373, R1509 (HEPN), H1514 (HEPN), Y1543, D1544,K1546, K1548, V1551, I1558, according to C2c2 consensus numbering. Incertain embodiments, the one or more modified of mutated amino acidresidues are one or more of those in C2c2 corresponding to R717 andR1509. In certain embodiments, the one or more modified of mutated aminoacid residues are one or more of those in C2c2 corresponding to K2, K39,K535, K1261, R1362, R1372, K1546 and K1548. In certain embodiments, saidmutations result in a protein having an altered or modified activity. Incertain embodiments, said mutations result in a protein having anincreased activity, such as an increased specificity. In certainembodiments, said mutations result in a protein having a reducedactivity, such as reduced specificity. In certain embodiments, saidmutations result in a protein having no catalytic activity (i.e. “dead”C2c2). In an embodiment, said amino acid residues correspond to Lsh C2c2amino acid residues, or the corresponding amino acid residues of a C2c2protein from a different species.

The invention also provides for the one or more mutations or the two ormore mutations to be in a catalytically active domain of the effectorprotein. In some embodiments of the invention the catalytically activedomain may comprise a RuvCI, RuvCII or RuvCIII domain, or acatalytically active domain which is homologous to a RuvCI, RuvCII orRuvCIII domain etc or to any relevant domain as described in any of theherein described methods. In certain embodiments, the one or moremutations or the two or more mutations may be in a catalytically activedomain of the effector protein comprising a HEPN domain, or acatalytically active domain which is homologous to a HEPN domain. Theeffector protein may comprise one or more heterologous functionaldomains. The one or more heterologous functional domains may compriseone or more nuclear localization signal (NLS) domains. The one or moreheterologous functional domains may comprise at least two or more NLSdomains. The one or more NLS domain(s) may be positioned at or near orin proximity to a terminus of the effector protein (e.g., C2c2) and iftwo or more NLSs, each of the two may be positioned at or near or inproximity to a terminus of the effector protein (e.g., C2c2). The one ormore heterologous functional domains may comprise one or moretranslational activation domains. In other embodiments the functionaldomain may comprise a transcriptional activation domain, for exampleVP64. The one or more heterologous functional domains may comprise oneor more transcriptional repression domains. In certain embodiments thetranscriptional repression domain comprises a KRAB domain or a SIDdomain (e.g. SID4X). The one or more heterologous functional domains maycomprise one or more nuclease domains. In a preferred embodiment anuclease domain comprises Fok1.

The invention also provides for the one or more heterologous functionaldomains to have one or more of the following activities: methylaseactivity, demethylase activity, transcription activation activity,transcription repression activity, transcription release factoractivity, histone modification activity, nuclease activity,single-strand RNA cleavage activity, double-strand RNA cleavageactivity, single-strand DNA cleavage activity, double-strand DNAcleavage activity and nucleic acid binding activity. At least one ormore heterologous functional domains may be at or near theamino-terminus of the effector protein and/or wherein at least one ormore heterologous functional domains is at or near the carboxy-terminusof the effector protein. The one or more heterologous functional domainsmay be fused to the effector protein. The one or more heterologousfunctional domains may be tethered to the effector protein. The one ormore heterologous functional domains may be linked to the effectorprotein by a linker moiety.

The invention also provides for the effector protein comprising aneffector protein from an organism from a genus comprising Streptococcus,Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia,Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta,Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter,Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium,Leptotrichia, Francisella, Legionella, Alicyclobacillus,Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes,Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae,Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium orAcidaminococcus. The effector protein may comprise a chimeric effectorprotein comprising a first fragment from a first effector proteinortholog and a second fragment from a second effector protein ortholog,and wherein the first and second effector protein orthologs aredifferent. At least one of the first and second effector proteinorthologs may comprise an effector protein from an organism comprisingStreptococcus, Campylobacter, Nitratifractor, Staphylococcus,Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum,Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium,Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae,Clostridiaridium, Leptotrichia, Francisella, Legionella,Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella,Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum,Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium orAcidaminococcus.

In certain embodiments, the effector protein, particularly a Type V locieffector protein, more particularly a Type V-B loci effector protein,even more particularly a C2c1p, may originate from, may be isolated fromor may be derived from a bacterial species belonging to the taxaBacilli, Verrucomicrobia, alpha-proteobacteria or delta-proteobacteria.In certain embodiments, the effector protein, particularly a Type V locieffector protein, more particularly a Type V-B loci effector protein,even more particularly a C2c1p, may originate from, may be isolated fromor may be derived from a bacterial species belonging to a genus selectedfrom the group consisting of Alicyclobacillus, Desulfovibrio,Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacillus,Desulfatirhabdium, Citrobacter, and Methylobacterium. In certainembodiments, the effector protein, particularly a Type V loci effectorprotein, more particularly a Type V-B loci effector protein, even moreparticularly a C2c1p, may originate, may be isolated or may be derivedfrom a bacterial species selected from the group consisting ofAlicyclobacillus acidoterrestris (e.g., ATCC 49025), Alicyclobacilluscontaminans (e.g., DSM 17975), Desulfovibrio inopinatus (e.g., DSM10711), Desulfonatronum thiodismutans (e.g., strain MLF-1), Opitutaceaebacterium TAV5, Tuberibacillus calidus (e.g., DSM 17572), Bacillusthermoamylovorans (e.g., strain B4166), Brevibacillus sp. CF 112,Bacillus sp. NSP2.1, Desulfatirhabdium butyrativorans (e.g., DSM 18734),Alicyclobacillus herbarius (e.g., DSM 13609), Citrobacter freundii(e.g., ATCC 8090), Brevibacillus agri (e.g., BAB-2500), Methylobacteriumnodulans (e.g., ORS 2060). In certain embodiments, the effector protein,particularly a Type V loci effector protein, more particularly a TypeV-B loci effector protein, even more particularly a C2c1p, mayoriginate, may be isolated or may be derived from a bacterial speciesselected from the group consisting of the bacterial species listed inthe Table in FIG. 41A-B.

In certain embodiments, the effector protein, particularly a Type V locieffector protein, more particularly a Type V-B loci effector protein,even more particularly a C2c1p, may comprise, consist essentially of orconsist of an amino acid sequence selected from the group consisting ofamino acid sequences shown in the multiple sequence alignment in FIG.13D-1-H-2.

In certain embodiments, a Type V-B locus as intended herein may encode aCas1-Cas4 fusion, Cas2, and the C2c1p effector protein. In certainembodiments, a Type V-B locus as intended herein may be adjacent to aCRISPR array. See FIG. 9 and FIG. 41A-B for illustration ofrepresentative Type V-B loci organization.

In certain embodiments, a Cas1 protein encoded by a Type V-B locus asintended herein may cluster with Type I-U system. See FIGS. 10A and 10Band FIG. 10C-1-10W illustrating a Cas1 tree including Cas1 encoded byrepresentative Type V-B loci.

In certain embodiments, the effector protein, particularly a Type V locieffector protein, more particularly a Type V-B loci effector protein,even more particularly a C2c1p, such as a native C2c1p, may be about1100 to about 1500 amino acids long, e.g., about 1100 to about 1200amino acids long, or about 1200 to about 1300 amino acids long, or about1300 to about 1400 amino acids long, or about 1400 to about 1500 aminoacids long, e.g., about 1100, about 1200, about 1300, about 1400 orabout 1500 amino acids long.

In certain embodiments, the effector protein, particularly a Type V locieffector protein, more particularly a Type V-B loci effector protein,even more particularly a C2c1p, and preferably the C-terminal portion ofsaid effector protein, comprises the three catalytic motifs of theRuvC-like nuclease (i.e., RuvCI, RuvCII and RuvCIII). In certainembodiments, said effector protein, and preferably the C-terminalportion of said effector protein, may further comprise a regioncorresponding to the bridge helix (also known as arginine-rich cluster)that in Cas9 protein is involved in crRNA-binding. In certainembodiments, said effector protein, and preferably the C-terminalportion of said effector protein, may further comprise a Zn fingerregion, which may be inactive (i.e., which does not bind zinc, e.g., inwhich the Zn-binding cysteine residue(s) are missing). In certainembodiments, said effector protein, and preferably the C-terminalportion of said effector protein, may comprise the three catalyticmotifs of the RuvC-like nuclease (i.e., RuvCI, RuvCII and RuvCIII), theregion corresponding to the bridge helix, and the Zn finger region,preferably in the following order, from N to C terminus: RuvCI-bridgehelix-RuvCII-Zinc finger-RuvCIII. See FIG. 11, FIG. 12-1-12-2 and FIGS.13A-1-13A-2 and 13C-1-13C-2 for illustration of representative Type V-Beffector proteins domain architecture.

In certain embodiments, Type V-B loci as intended herein may compriseCRISPR repeats between 30 and 40 bp long, more typically between 34 and38 bp long, even more typically between 36 and 37 bp long, e.g., 30, 31,32, 33, 34, 35, 36, 37, 38, 39, or 40 bp long.

In certain embodiments, the effector protein, particularly a Type V locieffector protein, more particularly a Type V-C loci effector protein,even more particularly a C2c3p, may originate, may be isolated or may bederived from a bacterial metagenome selected from the group consistingof the bacterial metagenomes listed in the Table in FIG. 43A-B.

In certain embodiments, the effector protein, particularly a Type V locieffector protein, more particularly a Type V-C loci effector protein,even more particularly a C2c3p, may comprise, consist essentially of orconsist of an amino acid sequence selected from the group consisting ofamino acid sequences shown in the multiple sequence alignment in FIG.13I-1-13I-4.

In certain embodiments, a Type V-C locus as intended herein may encodeCas1 and the C2c3p effector protein. See FIG. 14 and FIG. 43A-B forillustration of representative Type V-C loci organization.

In certain embodiments, a Cas1 protein encoded by a Type V-C locus asintended herein may cluster with Type I-B system. See FIGS. 10A and 10Band FIG. 10C-1-W illustrating a Cas1 tree including Cas1 encoded byrepresentative Type V-C loci.

In certain embodiments, the effector protein, particularly a Type V locieffector protein, more particularly a Type V-C loci effector protein,even more particularly a C2c3p, such as a native C2c3p, may be about1100 to about 1500 amino acids long, e.g., about 1100 to about 1200amino acids long, or about 1200 to about 1300 amino acids long, or about1300 to about 1400 amino acids long, or about 1400 to about 1500 aminoacids long, e.g., about 1100, about 1200, about 1300, about 1400 orabout 1500 amino acids long, or at least about 1100, at least about1200, at least about 1300, at least about 1400 or at least about 1500amino acids long.

In certain embodiments, the effector protein, particularly a Type V locieffector protein, more particularly a Type V-C loci effector protein,even more particularly a C2c3p, and preferably the C-terminal portion ofsaid effector protein, comprises the three catalytic motifs of theRuvC-like nuclease (i.e., RuvCI, RuvCII and RuvCIII). In certainembodiments, said effector protein, and preferably the C-terminalportion of said effector protein, may further comprise a regioncorresponding to the bridge helix (also known as arginine-rich cluster)that in Cas9 protein is involved in crRNA-binding. In certainembodiments, said effector protein, and preferably the C-terminalportion of said effector protein, may further comprise a Zn fingerregion. Preferably, the Zn-binding cysteine residue(s) may be conservedin C2c3p. In certain embodiments, said effector protein, and preferablythe C-terminal portion of said effector protein, may comprise the threecatalytic motifs of the RuvC-like nuclease (i.e., RuvCI, RuvCII andRuvCIII), the region corresponding to the bridge helix, and the Znfinger region, preferably in the following order, from N to C terminus:RuvCI-bridge helix-RuvCII-Zinc finger-RuvCIII. See FIGS. 13A-1-13A-2 and13C-1-13C-2 for illustration of representative Type V-C effectorproteins domain architecture. In particular embodiments, said effectorprotein may comprise two HEPN catalytic motifs as illustrated in FIG.97(A).

In certain embodiments, Type V-C loci as intended herein may compriseCRISPR repeats between 20 and 30 bp long, more typically between 22 and27 bp long, yet more typically 25 bp long, e.g., 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 bp long.

In certain embodiments, the effector protein, particularly a Type VIloci effector protein, more particularly a C2c2p, may originate from,may be isolated from, or may be derived from a bacterial speciesbelonging to the taxa alpha-proteobacteria, Bacilli, Clostridia,Fusobacteria and Bacteroidetes. In certain embodiments, the effectorprotein, particularly a Type VI loci effector protein, more particularlya C2c2p, may originate from, may be isolated from, or may be derivedfrom a bacterial species belonging to a genus selected from the groupconsisting of Lachnospiraceae, Clostridium, Carnobacterium,Paludibacter, Listeria, Leptotrichia, and Rhodobacter. In certainembodiments, the effector protein, particularly a Type VI loci effectorprotein, more particularly a C2c2p may originate from, may be isolatedfrom or may be derived from a bacterial species selected from the groupconsisting of Lachnospiraceae bacterium MA2020, Lachnospiraceaebacterium NK4A179, Clostridium aminophilum (e.g., DSM 10710),Lachnospiraceae bacterium NK4A144, Carnobacterium gallinarum (e.g., DSM4847 strain MT44), Paludibacter propionicigenes (e.g., WB4), Listeriaseeligeri (e.g., serovar ½b str. SLCC3954), Listeria weihenstephanensis(e.g., FSL R9-0317 c4), Listeria newyorkensis (e.g., strain FSLM6-0635), Leptotrichia wadei (e.g., F0279), Leptotrichia buccalis (e.g.,DSM 1135), Leptotrichia sp. Oral taxon 225 (e.g., str. F0581),Leptotrichia sp. Oral taxon 879 (e.g., strain F0557), Leptotrichiashahii (e.g., DSM 19757), Rhodobacter capsulatus (e.g., SB 1003, R121,or DE442). In certain embodiments, the effector protein, particularly aType VI loci effector protein, more particularly a C2c2p may originatefrom, may be isolated from or may be derived from a bacterial speciesselected from the group consisting of the bacterial species listed inthe Table in FIG. 42A-B. In particular embodiments, the C2c2 proteinoriginates from Leptotrichia shahii (e.g., DSM 19757).

In certain embodiments, the effector protein, particularly a Type VIloci effector protein, more particularly a C2c2p, may comprise, consistessentially of or consist of an amino acid sequence selected from thegroup consisting of amino acid sequences shown in the multiple sequencealignment in FIG. 13J-1-N-4 or more particularly from the groupconsisting of amino acid sequences shown in the sequence alignment inFIG. 110.

In certain embodiments, a Type VI locus as intended herein may encodeCas1, Cas2, and the C2c2p effector protein. In certain embodiments, aType V-C locus as intended herein may comprise a CRISPR array. Incertain embodiments, a Type V-C locus as intended herein may comprisethe c2c2 gene and a CRISPR array, and not comprise cas1 and cas2 genes.See FIG. 15 and FIG. 42A-B for illustration of representative Type VIloci organization.

In certain embodiments, a Cas1 protein encoded by a Type VI locus asintended herein may cluster within the Type II subtree along with asmall Type III-A branch, or within Type III-A system. See FIGS. 10A and10B and FIG. 10C-1-W illustrating a Cas1 tree including Cas1 encoded byrepresentative Type VI loci.

In certain embodiments, the effector protein, particularly a Type VIloci effector protein, more particularly a C2c2p, such as a nativeC2c2p, may be about 1000 to about 1500 amino acids long, such as about1100 to about 1400 amino acids long, e.g., about 1000 to about 1100,about 1100 to about 1200 amino acids long, or about 1200 to about 1300amino acids long, or about 1300 to about 1400 amino acids long, or about1400 to about 1500 amino acids long, e.g., about 1000, about 1100, about1200, about 1300, about 1400 or about 1500 amino acids long.

In certain embodiments, the effector protein, particularly a Type VIloci effector protein, more particularly a C2c2p, comprises at least oneand preferably at least two, such as more preferably exactly two,conserved RxxxxH motifs. Catalytic RxxxxH motifs are are characteristicof HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding) domains.Hence, in certain embodiments, the effector protein, particularly a TypeVI loci effector protein, more particularly a C2c2p, comprises at leastone and preferably at least two, such as more preferably exactly two,HEPN domains. See FIG. 11 and FIG. 13B and FIG. 110 for illustration ofrepresentative Type VI effector proteins domain architecture. In certainembodiments, the HEPN domains may possess RNAse activity. In otherembodiments, the HEPN domains may possess DNAse activity.

In certain embodiments, Type VI loci as intended herein may compriseCRISPR repeats between 30 and 40 bp long, more typically between 35 and39 bp long, e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 bp long.In particular embodiments, the direct repeat is at least 25 nt long.

In certain embodiments, a protospacer adjacent motif (PAM) or PAM-likemotif directs binding of the effector protein complex as disclosedherein to the target locus of interest. In some embodiments, the PAM maybe a 5′ PAM (i.e., located upstream of the 5′ end of the protospacer).In other embodiments, the PAM may be a 3′ PAM (i.e., located downstreamof the 5′ end of the protospacer). The term “PAM” may be usedinterchangeably with the term “PFS” or “protospacer flanking site” or“protospacer flanking sequence”.

In a preferred embodiment, the effector protein, particularly a Type Vloci effector protein, more particularly a Type V-B loci effectorprotein, even more particularly a C2c1p, may recognize a 5′ PAM. Incertain embodiments, the effector protein, particularly a Type V locieffector protein, more particularly a Type V-B loci effector protein,even more particularly a C2c1p, may recognize a 5′ PAM which is 5′ TTNor 5′ ATTN, where N is A, C, G or T. In certain preferred embodiments,the effector protein may be Alicyclobacillus acidoterrestris C2c1p, morepreferably Alicyclobacillus acidoterrestris ATCC 49025 C2c1p, and the 5′PAM is 5′ TTN, where N is A, C, G or T, more preferably where N is A, Gor T. In other preferred embodiments, the effector protein is Bacillusthermoamylovorans C2c1p, more preferably Bacillus thermoamylovoransstrain B4166 C2c1p, and the 5′ PAM is 5′ ATTN, where N is A, C, G or T.

In a preferred embodiment, the effector protein, particularly a Type VIloci effector protein, more particularly a C2c2p, may recognize a 3′PAM. In certain embodiments, the effector protein, particularly a TypeVI loci effector protein, more particularly a C2c2p, may recognize a 3′PAM which is 5′H, wherein H is A, C or U. In certain preferredembodiments, the effector protein may be Leptotrichia shahii C2c2p, morepreferably Leptotrichia shahii DSM 19757 C2c2, and the 5′ PAM is a 5′ H.

In certain embodiments, the CRISPR enzyme is engineered and can compriseone or more mutations that reduce or eliminate a nuclease activity.Mutations can also be made at neighboring residues, e.g., at amino acidsnear those indicated above that participate in the nuclease activity. Insome embodiments, only one HEPN domain is inactivated, and in otherembodiments, a second HEPN domain is inactivated.

In certain embodiments of the invention, the guide RNA or mature crRNAcomprises, consists essentially of, or consists of a direct repeatsequence and a guide sequence or spacer sequence. In certainembodiments, the guide RNA or mature crRNA comprises, consistsessentially of, or consists of a direct repeat sequence linked to aguide sequence or spacer sequence. In certain embodiments the guide RNAor mature crRNA comprises 19 nts of partial direct repeat followed by18, 19, 20, 21, 22, 23, 24, 25, or more nt of guide sequence, such as18-25, 19-25, 20-25, 21-25, 22-25, or 23-25 nt of guide sequence orspacer sequence. In certain embodiments, the effector protein is a C2c2effector protein and requires at least 16 nt of guide sequence toachieve detectable DNA cleavage and a minimum of 17 nt of guide sequenceto achieve efficient DNA cleavage in vitro. In particular embodiments,the effector protein is a C2c2 protein and requires at least 19 nt ofguide sequence to achieve detectable RNA cleavage. In certainembodiments, the direct repeat sequence is located upstream (i.e., 5′)from the guide sequence or spacer sequence. In a preferred embodimentthe seed sequence (i.e. the sequence essential critical for recognitionand/or hybridization to the sequence at the target locus) of the C2c2guide RNA is approximately within the first 5 nt on the 5′ end of theguide sequence or spacer sequence.

In preferred embodiments of the invention, the mature crRNA comprises astem loop or an optimized stem loop structure or an optimized secondarystructure. In preferred embodiments the mature crRNA comprises a stemloop or an optimized stem loop structure in the direct repeat sequence,wherein the stem loop or optimized stem loop structure is important forcleavage activity. In certain embodiments, the mature crRNA preferablycomprises a single stem loop. In certain embodiments, the direct repeatsequence preferably comprises a single stem loop. In certainembodiments, the cleavage activity of the effector protein complex ismodified by introducing mutations that affect the stem loop RNA duplexstructure. In preferred embodiments, mutations which maintain the RNAduplex of the stem loop may be introduced, whereby the cleavage activityof the effector protein complex is maintained. In other preferredembodiments, mutations which disrupt the RNA duplex structure of thestem loop may be introduced, whereby the cleavage activity of theeffector protein complex is completely abolished.

In particular embodiments, the C2c2 protein is an Lsh C2c2 effectorprotein and the mature crRNA comprises a stem loop or an optimized stemloop structure. In particular embodiments, the direct repeat of thecrRNA comprises at least 25 nucleotides comprising a stem loop. Inparticular embodiments, the the stem is amenable to individual baseswaps but activity is disrupted by most secondary structure changes ortruncation of the crRNA. Examples of disrupting mutations includeswapping of more than two of the stem nucleotides, addition of anon-pairing nucleotide in the stem, shortening of the stem (by removalof one of the pairing nucleotides) or extending the stem (by addition ofone set of pairing nucleotides). However, the crRNA may be amenable to5′ and/or 3′ extensions to include non-functional RNA sequences asenvisaged for particular applications described herein.

The invention also provides for the nucleotide sequence encoding theeffector protein being codon optimized for expression in a eukaryote oreukaryotic cell in any of the herein described methods or compositions.In an embodiment of the invention, the codon optimized nucleotidesequence encoding the effector protein encodes any C2c2 discussed hereinand is codon optimized for operability in a eukaryotic cell or organism,e.g., such cell or organism as elsewhere herein mentioned, for instance,without limitation, a yeast cell, or a mammalian cell or organism,including a mouse cell, a rat cell, and a human cell or non-humaneukaryote organism, e.g., plant.

In certain embodiments of the invention, at least one nuclearlocalization signal (NLS) is attached to the nucleic acid sequencesencoding the C2c2 effector proteins. In preferred embodiments at leastone or more C-terminal or N-terminal NLSs are attached (and hencenucleic acid molecule(s) coding for the C2c2 effector protein caninclude coding for NLS(s) so that the expressed product has the NLS(s)attached or connected). In certain embodiments of the invention, atleast one nuclear export signal (NES) is attached to the nucleic acidsequences encoding the C2c2 effector proteins. In preferred embodimentsat least one or more C-terminal or N-terminal NESs are attached (andhence nucleic acid molecule(s) coding for the C2c2 effector protein caninclude coding for NES(s) so that the expressed product has the NES(s)attached or connected). In a preferred embodiment a C-terminal and/orN-terminal NLS or NES is attached for optimal expression and nucleartargeting in eukaryotic cells, preferably human cells. In a preferredembodiment, the codon optimized effector protein is C2c2 and the spacerlength of the guide RNA is from 15 to 35 nt. In certain embodiments, thespacer length of the guide RNA is at least 16 nucleotides, such as atleast 17 nucleotides, preferably at least 18 nt, such as preferably atleast 19 nt, at least 20 nt, at least 21 nt, or at least 22 nt. Incertain embodiments, the spacer length is from 15 to 17 nt, from 17 to20 nt, from 20 to 24 nt, eg. 20, 21, 22, 23, or 24 nt, from 23 to 25 nt,e.g., 23, 24, or 25 nt, from 24 to 27 nt, from 27-30 nt, from 30-35 nt,or 35 nt or longer. In certain embodiments of the invention, the codonoptimized effector protein is C2c2 and the direct repeat length of theguide RNA is at least 16 nucleotides. In certain embodiments, the codonoptimized effector protein is C2c2 and the direct repeat length of theguide RNA is from 16 to 20 nt, e.g., 16, 17, 18, 19, or 20 nucleotides.In certain preferred embodiments, the direct repeat length of the guideRNA is 19 nucleotides.

The invention also encompasses methods for delivering multiple nucleicacid components, wherein each nucleic acid component is specific for adifferent target locus of interest thereby modifying multiple targetloci of interest. The nucleic acid component of the complex may compriseone or more protein-binding RNA aptamers. The one or more aptamers maybe capable of binding a bacteriophage coat protein. The bacteriophagecoat protein may be selected from the group comprising Qβ, F2, GA, fr,JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP,FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1. Ina preferred embodiment the bacteriophage coat protein is MS2. Theinvention also provides for the nucleic acid component of the complexbeing 30 or more, 40 or more or 50 or more nucleotides in length.

Accordingly, it is an object of the invention not to encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product. It may be advantageous in thepractice of the invention to be in compliance with Art. 53(c) EPC andRule 28(b) and (c) EPC. Nothing herein is to be construed as a promise.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

In a further aspect, the invention provides a eukaryotic cell comprisinga nucleotide sequence encoding the CRISPR system described herein whichensures the generation of a modified target locus of interest, whereinthe target locus of interest is modified according to in any of theherein described methods. A further aspect provides a cell line of saidcell. Another aspect provides a multicellular organism comprising one ormore said cells.

In certain embodiments, the modification of the target locus of interestmay result in: the eukaryotic cell comprising altered expression of atleast one gene product; the eukaryotic cell comprising alteredexpression of at least one gene product, wherein the expression of theat least one gene product is increased; the eukaryotic cell comprisingaltered expression of at least one gene product, wherein the expressionof the at least one gene product is decreased; or the eukaryotic cellcomprising an edited genome.

In certain embodiments, the eukaryotic cell may be a mammalian cell or ahuman cell.

In further embodiments, the non-naturally occurring or engineeredcompositions, the vector systems, or the delivery systems as describedin the present specification may be used for RNA sequence-specificinterference, RNA sequence specific modulation of expression (inludingisoform specific expression), stability, localization, functionality(e.g. ribosomal RNAs or miRNAs), etc; or multiplexing of such processes.

In further embodiments, the the non-naturally occurring or engineeredcompositions, the vector systems, or the delivery systems as describedin the present specification may be used for RNA detection and/orquantification within a cell.

In further embodiments, the the non-naturally occurring or engineeredcompositions, the vector systems, or the delivery systems as describedin the present specification may be used for generating disease modelsand/or screening systems.

In further embodiments, the non-naturally occurring or engineeredcompositions, the vector systems, or the delivery systems as describedin the present specification may be used for: site-specifictranscriptome editing or purturbation; nucleic acid sequence-specificinterference; or multiplexed genome engineering.

Also provided is a gene product from the cell, the cell line, or theorganism as described herein. In certain embodiments, the amount of geneproduct expressed may be greater than or less than the amount of geneproduct from a cell that does not have altered expression or editedgenome. In certain embodiments, the gene product may be altered incomparison with the gene product from a cell that does not have alteredexpression or edited genome.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIGS. 1A-1B depicts a new classification of CRISPR-Cas systems. Class 1includes multisubunit crRNA-effector complexes (Cascade) and Class 2includes Single-subunit crRNA-effector complexes (Cas9-like). FIG. 1Bprovides another depiction of the new classification of CRISPR-Cassystems.

FIG. 2 provides a molecular organization of CRISPR-Cas.

FIGS. 3A-3D provides structures of Type I and III effector complexes:common architecture/common ancestry despite extensive sequencedivergence.

FIG. 4 shows CRISPR-Cas as a RNA recognition motif (RRM)-centeredsystem.

FIG. 5 shows a Cas1 phylogeny where recombination of adaptation andcrRNA-effector modules show a major aspect of CRISPR-Cas evolution.

FIG. 6 shows a CRISPR-Cas census, specifically a distribution ofCRISPR-Cas types/subtypes among archaea and bacteria.

FIG. 7 depicts a pipeline for identifying Cas candidates.

FIGS. 8A-8B depicts an organization of complete loci of Class 2CRISPR-Cas systems. The three subtypes of type II and subtypes V-A, V-Band V-C, and type VI are indicated. Subfamilies based on Cas1 are alsoindicated. The schematics include only the common genes represented ineach subtype; the additional genes present in some variants are omitted.The red rectangle shows the degenerate repeat. The gray arrows show thedirection of CRISPR array transcription. PreFran,Prevotella-Francisella. FIG. 8B provides another depiction of anorganization of complete loci of several Class 2 CRISPR-Cas systems.

FIG. 9 depicts C2c1 neighborhoods, i.e., genomic architecture of theC2c1 CRISPR-Cas loci. The number of repeats in CRISPR arrays isindicated. For each genomic contig, Genbank numeric ID and thecoordinates of the locus are indicated.

FIGS. 10A-10B. FIGS. 10A and 10B depict representations of a Cas1 tree.The tree in FIG. 10B was constructed from a multiple alignment of 1498Cas1 sequences which contained 304 phylogenetically informativepositions. Branches, corresponding to Class 2 systems are highlighted:cyan, type II; orange, subtype V-A; red, subtype V-B; brown, subtypeV-C; purple, type VI. Insets show the expanded branches of the novel(sub)types. The bootstrap support values are given as percentage pointsand shown only for few relevant branches.

FIGS. 10C-1-10W. FIGS. 10C-1-10W provide the complete Cas1 tree, whichis schematically shown in FIG. 10B, in Newick format with species namesand bootstrap support values. The tree was reconstructed by FastTreeprogram (“-gamma -wag” options). A multiple alignment of Cas1 sequenceswas filtered with homogeneity threshold of 0.1 and gap occurrencethreshold of 0.5, prior to tree reconstruction.

FIG. 11 depicts a domain organization of class 2 families.

FIG. 12-1-12-2 depicts TnpB homology regions in Class 2 proteins.

FIGS. 13A-1-13N-4. FIGS. 13A-1-A-2 and 13B provide another depiction ofdomain architectures and conserved motifs of the Class 2 effectorproteins. FIG. 13A-1-A-2 illustrates Types II and V: TnpB-derivednucleases. The top panel shows the RuvC nuclease from Thermosthermophilus (PDB ID: 4EP5) with the catalytic amino acid residuesdenoted. Underneath each domain architecture, an alignment of theconserved motifs in selected representatives of the respective proteinfamily (a single sequence for RuvC) is shown. The catalytic residues areshown by white letters on a black background; conserved hydrophobicresidues are highlighted in yellow; conserved small residues arehighlighted in green; in the bridge helix alignment, positively chargedresidues are in red. Secondary structure prediction is shown underneaththe aligned sequences: H denotes α-helix and E denotes extendedconformation (β-strand). The poorly conserved spacers between thealignment blocks are shown by numbers. FIG. 13B illustrates Type VI:proteins containing two HEPN domains, which may display RNAse activity.The top alignment blocks include selected HEPN domains describedpreviously and the bottom blocks include the catalytic motifs from thetype VI effector proteins. The designations are as in FIG. 13A-1-A-2.FIG. 13C-1-C-2 shows the closest homologs of the new type V effectorproteins among the transposon-encoded proteins: non-overlapping sets ofhomologs. FIG. 13D-1-H-2 shows multiple alignment of C2c1 proteinfamily. The alignment was built using MUSCLE program and modifiedmanually on the basis of local PSI-BLAST pairwise alignments. Eachsequence is labelled with GenBank Identifier (GI) number and systematicname of an organism. Secondary structure was predicted by Jpred andshown underneath the sequence which was used as a query (designations:H—alpha helix, E-beta strand). CONSENSUS was calculated for eachalignment column by scaling the sum-of-pairs score within the columnbetween those of a homogeneous column (the same residue in all alignedsequences) and a random column with homogeneity cutoff 0.8. Active sitemotifs of RuvC-like domain are shown below alignment. FIG. 13I-1-I-4shows multiple alignment of C2c3 protein family. The alignment was builtusing MUSCLE program. Each sequence is labelled with local assignednumber and the Genbank ID for metagenomics contig coding for respectiveC2c3 protein. Secondary structure was predicted by Jpred and shownunderneath the alignment (designations: H-alpha helix, E—beta strand).CONSENSUS was calculated for each alignment column by scaling thesum-of-pairs score within the column between those of a homogeneouscolumn (the same residue in all aligned sequences) and a random columnwith homogeneity cutoff 0.8. Active site motifs of RuvC-like domain areshown below alignment for the C-terminal domain. FIG. 13J-1-N-4 showsmultiple alignment of C2c2 protein family. The alignment was built usingMUSCLE program and modified manually on the basis of local PSIBLASTpairwise alignments. Each sequence is labelled with GenBank Identifier(GI) number and systematic name of an organism. Secondary structure waspredicted by Jpred and shown underneath the sequence which was used as aquery (designations: H—alpha helix, E—beta strand). CONSENSUS wascalculated for each alignment column by scaling the sum-of-pairs scorewithin the column between those of a homogeneous column (the sameresidue in all aligned sequences) and a random column with homogeneitycutoff 0.8. Active site motifs of HEPN domain are shown below alignment.

FIG. 14 depicts C2c3 neighborhoods, i.e., genomic architecture of theC2c3 CRISPR-Cas loci. The number of repeats in CRISPR arrays isindicated. For each genomic contig, Genbank numeric ID and thecoordinates of the locus are indicated.

FIG. 15 depicts C2c2 neighborhoods.

FIG. 16-1-16-8 depicts HEPN RxxxxH motif in C2c2 family.

FIG. 17 depicts C2C1: 1. Alicyclobacillus acidoterrestris ATCC 49025

FIG. 18 depicts C2C1: 4. Desulfonatronum thiodismutans strain MLF-1

FIG. 19 depicts C2C1: 5. Opitutaceae bacterium TAV5

FIG. 20 depicts C2C1: 7. Bacillus thermoamylovorans strain B4166

FIG. 21 depicts C2C1: 9. Bacillus sp. NSP2.1

FIG. 22 depicts C2C2: 1. Lachnospiraceae bacterium MA2020

FIG. 23-1-23-2 depicts C2C2: 2. Lachnospiraceae bacterium NK4A179

FIG. 24 depicts C2C2: 3. [Clostridium] aminophilum DSM 10710

FIG. 25 depicts C2C2: 4. Lachnospiraceae bacterium NK4A144

FIG. 26 depicts C2C2: 5. Carnobacterium gallinarum DSM 4847

FIG. 27-1-27-2 depicts C2C2: 6. Carnobacterium gallinarum DSM 4847

FIG. 28 depicts C2C2: 7. Paludibacter propionicigenes WB4

FIG. 29 depicts C2C2: 8. Listeria seeligeri serovar ½b

FIG. 30 depicts C2C2: 9. Listeria weihenstephanensis FSL R9-0317

FIG. 31-1-31-2 depicts C2C2: 10. Listeria bacterium FSL M6-0635

FIG. 32 depicts C2C2: 11. Leptotrichia wadei F0279

FIG. 33-1-33-2 depicts C2C2: 12. Leptotrichia wadei F0279

FIG. 34 depicts C2C2: 14. Leptotrichia shahii DSM 19757

FIG. 35 depicts C2C2: 15. Rhodobacter capsulatus SB 1003

FIG. 36 depicts C2C2: 16. Rhodobacter capsulatus R121

FIG. 37 depicts C2C2: 17. Rhodobacter capsulatus DE442

FIG. 38 depicts a tree of DRs

FIG. 39 depicts a tree of C2c2s

FIGS. 40A-40D shows the Table listing 63 large protein-coding genesidentified using the computational pipeline disclosed herein in thevicinity of cas1 genes. Representatives of the new subtypes disclosedherein (V-B, V-C, VI) are colored. Protein sequences forAUXO014641567.1, AUXO011689375.1, AUXO011689375.1, AUXO011277409.1,AUXO014986615.1 coding representatives of Type V-B and Type IV were notanalyzed, since species affiliation cannot be assigned to thesesequences.

FIGS. 41A-41M-2. FIGS. 41A-B shows the Table presenting the analysis ofType V-B (C2c1 protein-encoding) loci. * cas1cas4—gene containing cas4and cas1 domains; CRISPR—CRISPR repeat; SOS—SOS response gene;unk—hypothetical protein; >—direction of gene coding sequence;[D]—degenerate repeat (defined where it was possible); [T]-tracrRNA.FIG. 41C-J shows CRISPR arrays analysis of Type V-B (C2c1protein-encoding) loci as disclosed herein (CRISPR section is basicoutput of pilercr (see pilercr site for description of output:http://www.drive5.com/pilercr/); repeat folding was done with mfold (seemfold site for description of output:http.//mfold.rna.albany.edu/?q=mfold/DNA-Folding-Form); repeat foldingand CRISPRS array are placed after detailed description of each case;for CRISPR location see link in the Table in FIG. 41A-B). FIG. 41K showsCRISPRmap classification of CRISPR repeats of Type V-B (C2c1protein-encoding) loci as disclosed herein using CRISPRmap (seehttp://rna.informatik.uni-freiburg.de/CRISPRmap/Input.jsp for details).FIG. 41L shows degenerate repeats of Type V-B (C2c1 protein-encoding)loci as disclosed herein found using CRISPRs finder(http://crispr.u-psud.fr/Server/). Normal repeat column contains normalrepeat, spacer—the last spacer, downstream—downstream region startingfrom degenerate repeat (250 bp); array number corresponds to the numberof CRISPR array in the respective locus (see the Table in FIG. 41A-B);region highlighted in yellow has a perfect match between normal repeatand degenerate repeat (other part of degenerate repeat does not match).FIG. 41M-1-M-2 shows predicted structures of tracrRNAs base-paired withthe repeats. TracrRNA for Alicyclobacillus acidoterrestric was identifedusing RNAseq. For the remaining loci, putative tracrRNAs were identifedbased on presence of an anti-direct repeat (DR) sequence. Anti-DRs wereidentifed using Geneious (www.geneious.com) by searching for sequenceswithin each respective CRISPR locus that are highly homologus to DR. The5′ and 3′ ends of each putative tracrRNA was determined thoughcomputational prediction of bacterial transcription start andtermination sites using BPROM (www.softberry.com) and ARNOLD(rna.ig-mors.u-psud.fr/toolbox/arnold/) respectively. Co-foldingpredictions were generated using Geneious. 5′ ends are colored blue and3′ ends are colored orange.

FIGS. 42A-42N-2. FIG. 42A-B shows the Table presenting the analysis ofType VI (C2c2 protein-encoding) loci. * CRISPR—CRISPR repeat;unk—hypothetical protein; >—direction of gene coding sequence;[D]—degenerate repeat (defined where it was possible); [T]-tracrRNA.FIG. 42C-1-I-3 shows CRISPR arrays analysis of Type VI (C2c2protein-encoding) loci as disclosed herein (CRISPR section is basicoutput of pilercr (see pilercr site for description of output:http://www.drive5.com/pilercr/); repeat folding was done with mfold (seemfold site for description of output:http.//mfold.rna.albany.edu/?q=mfold/DNA-Folding-Form); repeat foldingand CRISPRS array are placed after detailed description of each case;for CRISPR location see link in the Table in FIG. 42A-B). FIG. 42J-1-J-2shows CRISPRmap classification of CRISPR repeats of Type VI (C2c2protein-encoding) loci as disclosed herein using CRISPRmap (seehttp://rna.informatik.uni-freiburg.de/CRISPRmap/Input.jsp for details).FIG. 42K-1-L shows degenerate repeats of Type VI (C2c2 protein-encoding)loci as disclosed herein found using CRISPRs finder(http://crispr.u-psud.fr/Server/). Normal repeat column contains normalrepeat, spacer—the last spacer, downstream—downstream region startingfrom degenerate repeat (250 bp); array number corresponds to the numberof CRISPR array in the respective locus (see the Table in FIG. 42A-B);region highlighted in yellow has a perfect match between normal repeatand degenerate repeat (other part of degenerate repeat does not match).FIG. 42M-N-2 shows predicted structures of tracrRNAs base-paired withthe repeats. Putative tracrRNAs were identifed based on presence of ananti-direct repeat (DR) sequence. Anti-DRs were identifed using Geneious(www.geneious.com) by searching for sequences within each respectiveCRISPR locus that are highly homologus to DR. The 5′ and 3′ ends of eachputative tracrRNA was determined though computational prediction ofbacterial transcription start and termination sites using BPROM(www.softberry.com) and ARNOLD (rna.ig-mors.u-psud.fr/toolbox/arnold/)respectively. Co-folding predictions were generated using Geneious. 5′ends are colored blue and 3′ ends are colored orange.

FIGS. 43A-43F. FIG. 43A-B shows the Table presenting the analysis ofType V-C (C2c3 protein-encoding) loci. * CRISPR—CRISPR repeat;unk—hypothetical protein; >—direction of gene coding sequence;[D]—degenerate repeat (defined where it was possible). FIG. 43C-D-2shows CRISPR arrays analysis of Type V-C(C2c3 protein-encoding) loci asdisclosed herein (CRISPR section is basic output of CRISPRfinder (seefor description: http://crispr.u-psud.fr/Server/); repeat folding wasdone with mfold (see mfold site for description of output:http://mfold.rna.albany.edu/?q=mfold/DNA-Folding-Form); repeat foldingand CRISPRS array are placed after detailed description of each case;for CRISPR location see link in the Table in FIG. 43A-B). Statisticallysignificant spacer's blast hits in prokaryotes or their viruses areshown. FIG. 43E shows CRISPRmap classification of CRISPR repeats of TypeV-C(C2c3 protein-encoding) loci as disclosed herein using CRISPRmap (seehttp://rna.informatik.uni-freiburg.de/CRISPRmap/Input.jsp for details).FIG. 43F shows degenerate repeats of Type V-C (C2c3 protein-encoding)loci as disclosed herein found using CRISPRs finder(http://crispr.u-psud.fr/Server/). Normal repeat column contains normalrepeat, spacer—the last spacer, downstream—downstream region startingfrom degenerate repeat (250 bp); array number corresponds to the numberof CRISPR array in the respective locus (see the Table in FIG. 43A-B);region highlighted in yellow has a perfect match between normal repeatand degenerate repeat (other part of degenerate repeat does not match).

FIGS. 44A-44E-2 provides complete list of CRISPR-Cas loci in the genomeswhere C2c1 or C2c2 proteins were found. Genes for C2c1 and C2c2 proteinsare highlighted in yellow.

FIGS. 45A-45C shows alignment of Listeria loci encoding putative Type VICRISPR-Cas system. The aligned syntenic region corresponds to Listeriaweihenstephanensis FSL R9-0317 contig AODJ01000004.1, coordinates42281-46274 and Listeria newyorkensis strain FSL M6-0635 contigJNFB01000012.1, coordinates 169489-173541. Color coding: C2c2 gene ishighlighted by blue CRISPR repeats—red, degenerated repeat—magenta,spacers—bold.

FIG. 46 shows the two C2c2 loci of Carnobacterium gallinarum.

FIG. 47 shows a schematic indicating the expression level of two CRISPRarrays in the direction of the C2c2 gene at the first C2c2 locus.

FIG. 48 shows a schematic indicating the expression level of CRISPRarrays with direction of transcription in the direction of the C2c2 geneat the second C2c2 locus.

FIGS. 49A-49B. FIGS. 49A-49B shows expression and processing of C2c2loci.

FIG. 49A: RNA-sequencing of the Listeria seeligeria serovar ½b str.SLCC3954 C2c2 locus expressed in E. coli. The locus is highly expressedwith a processed crRNA showing a 5′ 29-nt DR and 15-18nt spacer. Theputative tracrRNA shows no expression. In silico RNA-folding of theprocessed crRNA direct repeat shows a strong hairpin. FIG. 49B: Northernblot analysis of the Leptotrichia shahii DSM 19757 expressed in E. colishows processed crRNAs with a 5′ DR. Arrows indicate the probe positionsand their directionality.

FIGS. 50A-50C. FIGS. 50A-C shows expression and processing of theLeptotrichia shahii DSM 19757 C2c2 locus. FIG. 50A: RNA-sequencing ofthe Leptotrichia shahii DSM 19757 locus expressed in E. coli showsprocessing of the CRISPR array in the 3′ to 5′ direction (direction ofthe locus). crRNAs are processed to have a 5′ DR that is 28nt in lengthand spacers with lengths 14-28nt. FIG. 50B RNA-sequencing of theendogenous Leptotrichia shahii DSM 19757 C2c2 locus shows similarresults to FIG. 50A. FIG. 50C: In silico folding of the L. shahii crRNADR predicts stable secondary structure.

FIG. 51 shows evolutionary scenario for the CRISPR-Cas systems. The Cas8protein is hypothesized to have evolved by inactivation of Cas10 (shownby the white X) which was accompanied by a major acceleration ofevolution. Abbreviations: TR, terminal repeats; TS, terminal sequences;HD, HD family endonuclease; HNH, HNH family endonuclease; RuvC, RuvCfamily endonuclease; HEPN, putative endoribonuclease of HEPNsuperfamily. Genes and protein regions shown in gray denote sequencesthat were encoded in the respective mobile elements but were eliminatedin the course of evolution of CRISPR-Cas systems.

FIG. 52 depicts arrangement of C2c2 gene locus, including domainsbelonging to the HEPN domain superfamily. The majority of HEPN domainscontain conserved motifs and constitute metal-independent endoRNases.

FIG. 53 depicts RNAseq analysis of an endogenous locus from Leptotrichiashahii DSM 19757.

FIG. 54 is a cartoon depicting in vivo experiment using E. coliexpressing LshC2c2 to identify depleted sequence motifs. A PAM libraryconferring ampicillin resistance is transferred into E. coli. Plasmidscarrying sequence motifs containing a PAM determinant are lost andunable to confer ampicillin resistance. PAM sequence motifs areidentified by their depletion.

FIG. 55 Identification of PAM sequence. Depleted sequences identify the5′ PAM nucleotides

FIG. 56 depicts targeting of an endogenous target in E. coli.Interference is indicated by a reduction in colony forming units (cfu)pre 20 ng of plasmid. Interference was observed in E. coli carryingLshC2C but not with a control pACYC184. Increased interference isassociated with a transcribed target.

FIG. 57 depicts purification of C2c2 by His-Tag purification followed byone round (left) or three rounds (middle) of gel filtration, and asingle 168 kD band by coomassie stain (right).

FIG. 58 depicts components of in vitro experiments with purified LshC2c2 and FPLC purified RNA target. Component “166” indicates anon-complementary target. “E” indicate EDTA. Cleavage of crRNA isobserved when present with C2c2.

FIG. 59 depicts in vitro experiments with purified C2c2, RNA target andcrRNAs with spacer lengths of 12-26 nucleotides. Cleavage of crRNAs with28 or 24 nt spacers is observed.

FIG. 60 depicts an electrophoretic mobility shift assay (EMSA) useful todetect protein complexes with nucleic acids.

FIG. 61 depicts targeting in vivo of transcribed red fluorescent protein(RFP) using RFP spacers matching or complementary to RNA. Spacerstargeting transcribed RFP were cloned into the Lsh C2c2 locus followedby expression in E. coli carrying a plasmid expressing RFP or a pUC19plasmid control. Interference was determined on the basis of colonyforming units (cfu) per 20 ng of transformed plasmid.

FIG. 62 depicts strand-dependent interference with plasmid carrying anRFP target. Interference was measured as colony forming units (cfu) for6 RFP targets (left panel). Interference was observed to depend on thestrand targeted and the PAM nucleotide present. Interference withnon-targeted pUC19 control plasmid was not observed (right panel).

FIGS. 63A-63C. FIG. 63 depicts effect on RFP expression of C2c2 withtargeting RNA complementary or non-complementary to expressed RNA. ASchematic of RFP targeting in heterologous E. coli system. LshC2c2 lociharboring spacers targeting RFP at various PAMs were introduced intoRFP-expressing E. coli. B Quantitation of RFP targeting in E. coli formultiple spacers targeting A, C, or U PAMs. RFP expression was measuredby flow cytometry. C Quantitation of RFP targeting in E. coli. Spacerswith various PAMs targeting either the non-coding strand (“DNA”) orcoding strand (“RNA”) of the RFP gene were introduced and RFP expressionwas measured by flow cytometry.

FIG. 64 depicts processing of direct repeat (DR) sequences by LshC2c2.LshC2c2 processes the DR on the 5′ end.

FIG. 65 depicts strategy for investigating target site selection. Target1 (T1) contains a G PAM, Target 3 (T3) contains a C PAM.

FIG. 66 depicts results of a C2c2 RNA cleavage reaction targeted to T3(see FIG. 65). Reaction components are indicated.

FIG. 67 depicts results of a C2c2 RNA cleavage reaction targeted to T1(see FIG. 65). Reaction components are indicated.

FIG. 68 depicts C2c2-mediated cleavage directed to RNA expressed from aDNA template in vitro. Reaction components are indicated. Lanes 2-8:C2c2-mediated cleavage is targeted to T1 (see FIG. 65). Lanes 9-15:C2c2-mediated cleavage is targeted to the reverse complement of T1.

FIG. 69 depicts C2c2-mediated cleavage directed to RNA expressed from aDNA template in vitro. Reaction components are indicated. Lanes 2-8:C2c2-mediated cleavage is targeted to T3 (see FIG. 65).

FIG. 70 depicts RNA fragment sizes observed for C2c2-mediated cleavagetargeted to T1 or T3 (see FIG. 65).

FIG. 71 depicts C2c2 mediated RNA cleavage of targets T1 and T3. Thereare multiple cleavage products and significant reduction in intensity ofthe target band. Buffer 1: 40 mM Tris-HCl (pH 7.9), 6 mM MgCL₂, 83 mMNaCl, 1 mM DTT, rNTPs, T7 polymerase. Buffer 2: 25 mM Tris-HCl (pH 7.5),10 mM MgCL₂, 83 mM NaCl, 5 mM DTT. Buffer 2 replicates reactionconditions without DNA template.

FIG. 72 shows mutation of either HEPN domain abolishes RNA targeting.

FIG. 73 shows a schematic overview of an RNA PAM screen using MS2 phageinterference. A library consisting of spacers targeting all possiblesequences in the MS2 RNA genome was cloned into the LshC2c2 CRISPRarray. Cells with this library population were then treated with phageand plated, and surviving cells were harvested. Frequency of spacerswere compared to an phage-untreated control, and phage-enriched spacerswere used for generation of sequence logos.

FIG. 74 indicates that RNA phage interference screen shows both strongenrichment and depletion of LshC2c2 spacers by RNA PAM screen using MS2phage interference.

FIGS. 75A-75J. FIGS. 75A-C shows identification of a single base rightPAM for LshC2c2 by RNA PAM screen using MS2 phage interference. Moreparticularly it shows the presence of a right H PAM (not G). FIG. 75Cshows the quantitation of MS2 plaque assay validating the presence ofthe PAM. Multiple spacers targeting each PAM were cloned into theLshC2c2 locus. Phage dilutions were spotted on bacteria plates andinterference was quantified by highest dilution without plaques. FIG.75D shows representative images from validation of MS2 plaque assayshowing reduced plaque formation in H PAM spacers, but not in G PAMspacers. FIG. 75E shows nuclease activity with G PAM spacers andresistance with H PAM spacers. FIG. 75 F shows a schematic of the RNAtarget, showing the protospacer region and the corresponding crRNA. FIG.75 G shows denaturing gel demonstrating ssRNA cleavage activity ofLshC2c2 using an RNA target that is 5′ labeled with IRDye 800 and 3′labeled with Cy5. Four independent cleavage sites are observed. FIG. 75H shows a denaturing gel demonstrating the H PAM (not G). ssRNA cleavageactivity is dependent on the nucleotide immediately 3′ of the targetsite. The PAM was tested by mutating this adjacent nucleotide and usingthe same crRNA/target site. FIG. 75 I is a schematic showing thepositions of tiled crRNAs to demonstrate retargeting of LshC2c2 and theH PAM (top panel) and the corresponding denaturing gel (bottom panel).Five different crRNAs were tested for each possible nucleotide. Alsoshown is a denaturing gel demonstrating the H PAM using different crRNAstiled along the length of the ssRNA target. Five different crRNAs weretested for each possible nucleotide. FIG. 75J shows a denaturing geltesting the presence of a spacer motif. Three crRNAs with every possiblelast nucleotide as the last base of the spacer sequence were tested.

FIG. 76 shows for LshC2c2 RNA phage MS2 restriction. Cloned spacerstargeting each of the four MS2 genes, with C and G PAMs. G PAMs requirehigher phage concentrations for plaque development.

FIG. 77 shows that targeting of RFP transcripts in bacteria slows growthrate.

FIGS. 78A-78D. FIGS. 78 A-B demonstrates that LshC2c2 efficientlycleaves RNA. Mini gel readout; non fluorescence. FIG. 78C demonstratesssRNA cleavage by LshC2c2 using a 5′ labeled and 3′ labeled target wereassayed at the indicated time points. FIG. 78D shows the quantitation ofdata of FIG. 78B.

FIG. 79: LshC2c2 protein and crRNA were incubated and serially diluted.ssRNA cleavage was assayed using the indicated complex concentrations.

FIG. 80 demonstrates that LshC2c2 efficiently cleaves RNA. 20 cm gelreadout; 700 nm fluorescent imaging. Cleavage is observed just with asmaller 85nt RNA target instead of the usual 173nt target.

FIG. 81 shows mapping of cleavage fragments.

FIG. 82 shows RNA-sequencing of in vitro nuclease reaction

FIG. 83 demonstrates that LshC2c2 does not cleave untranscribed ortranscribed DNA in an E. coli RNAP in vitro assay. Assay set-up asdescribed in Samai et al. (Cell, 2015). 200 bp target is used(corresponding to RNA target of FIG. 82). 1 h incubation at 37° C. forconcurrent transcription and cleavage after open complex formation.

FIG. 84 demonstrates that LshC2c2 does not cleave ssDNA in vitro. 1 hincubation at 37° C., ssDNA version of t3 G-PAM and its reversecomplement (RC).

FIGS. 85A-85B. FIG. 85 demonstrates that C2c2 does not cleave dsDNA(FIG. 85A) and ssDNA (FIG. 85B). Band gel extracted and prepared fornext generation sequencing by Illumina MiSeq.

FIG. 86 demonstrates that LshC2c2 has a 3′ G PAM for RNA cleavage. Sametarget with varying PAMs. (PAM sequence shown is on reverse complement,such that 5′ C corresponds to 3′G).

FIG. 87 demonstrates that LshC2c2 does not require the small RNA for RNAcleavage.

FIG. 88 demonstrates that LshC2c2 is reprogrammable and PAM sensitive.

FIG. 89 demonstrates that LshC2c2 is reprogrammable and PAM sensitive.

FIGS. 90A-90B. FIGS. 90A and 90B demonsrate ssRNA cleavage was assayedusing crRNAs of varied spacer length.

FIGS. 91A-91C. FIGS. 91 A, 91B, and 91C demonstrates ssRNA cleavage wasassayed using crRNAs of varied DR length. RNA cleavage is crRNA lengthdependent (FIG. 91A, 91B). RNA cleavage is DR length dependent FIG.91C-91E).

FIGS. 92A-92D. FIGS. 92 A, 92B, 92C, and 92D show LshC2c2 cr StemModifications and demonstrates that Stem is amenable to individual baseswaps but activity is disrupted by most secondary structure changes. DRTruncation experiments also indicate that disruption of the stemabolishes cleavage.

FIGS. 93A-93D. FIGS. 93A, 93B, 93C, and 93D shows LshC2c2 cr loopModifications and demonstrates that the crRNA loop is amenable to basechanges and extension but not truncation

FIG. 94 demonstrates that C2c2 processes its own array . . . Buffer 1:40 mM Tris-HCl (pH 7.9), 6 mM MgCl2, 70 mM NaCl, 1 mM DTT. Buffer 2: 40mM Tris-HCl (pH 7.3), 6 mM MgCl2, 70 mM NaCl, 1 mM DTT, Murine RNAseinhibitor. LshC2c2 cleaves both array 1 and array 2 (arrays of differentlengths) in both buffers tested. Cleavage is evident by generation oflower bands

FIG. 95 demonstrates that HEPN mutants still process the c2c2 CRISPRarray.

FIG. 96 demonstrates that processing of the C2c2 array in E colirequires the C2c2 protein.

FIGS. 97A-97D. FIG. 97 A schematic shows different HEPN mutations inC2c2. Schematic of locus and LshC2c2 protein, showing conserved residuesin HEPN domains. FIGS. 97 B and 97C demonstrate that each of the HEPNmutants significantly lack activity. C top panel: Denaturing gel showingconserved residues of the HEPN motif are necessary for ssRNA cleavage. Cbottom panel: Quantitation of MS2 plaque assay with HEPN catalyticresidue mutants. Loci with mutant LshC2c2 were unable to protect againstphage. FIG. 97D. Quantitation of RFP targeting in E. coli with arginineHEPN catalytic residue mutants. Loci with mutant LshC2c2 were unable toknock down RFP.

FIG. 98 shows bystander effect. Once active, C2c2 seems to become activeand degrade other RNAs in the sample. Top panel: L=long target,small=small target, LC=long target with C PAM. Bottom panel: effect ofpresence or absence of activator target on cleavage of differentbystander targets.

FIGS. 99A-C. FIG. 99 A demonstratres that the C2c2 HEPN mutant stillbinds the crRNA. FIGS. 99B and 99C demonstrate the influence ofsecondary structure of the RNA on cleavage product of the target RNA

FIGS. 100A-100B. FIG. 100 A shows the effect of different divalentcations on C2c2 activity. FIG. 100 B shows the effect of crRNA titrationin the presence or absence of magnesium.

FIG. 101 shows a denaturing gel demonstrating ssRNA cleavage activity ofLshC2c2 using an RNA target that is 5′ labeled with IRDye 800 and 3′labeled with Cy5. Four independent cleavage sites are observed. Thisfigures also shows the effect of Mg++ chelation on ssRNA cleavageactivity.

FIG. 102 demonstrates that C2c2 cuts 3′ of the target site.

FIG. 103 demonstrates that C2c2 cuts 3′ of target site.

FIG. 104 shows C2c2 reprogramming with crRNAs.

FIG. 105 shows C2c2 reprogramming with crRNAs.

FIG. 106 shows C2c2 reprogramming with crRNAs.

FIG. 107 shows IVC (in vitro nuclease reaction) of a long target.

FIG. 108 shows bystander effect. Reduced cleavage is observed in absenceof magnesium.

FIG. 109-1-109-2 illustrates the sequences alignment of the followingorthologs of the Leptotrichia shahii DSM 19757 C2c2: Rhodobactercapsulatus SB 1003 (RcS); Rhodobacter capsulatus R121 (RcR); Rhodobactercapsulatus DE442 (RcD); Lachnospiraceae bacterium MA2020 (Lb(X));Lachnospiraceae bacterium NK4A179 (Lb(X); [Clostridium] aminophilumDSM_10710 (CaC); Lachnospiraceae bacterium NK4A144 (Lb(X); Leptotrichiawadei F0279 (Lew); Leptotrichia wadei F0279 (Lew); Carnobacteriumgallinarum DSM 4847 (Cg); Carnobacterium gallinarum DSM 4847 (Cg);Paludibacter propionicigenes WB4 (Pp); Listeria seeligeri serovar ½b(Ls); Listeria weihenstephanensis FSL R9-0317 (Liw); and Listeriabacterium FSL M6-0635 (Lib).

FIG. 110 depicts conserved HEPN domains of C2c2 proteins.

FIG. 111. Demonstrates that C2c2 HEPN mutains retain targeted bindingactivity. Top panels: electrophoretic mobility shift assay with wildtype LshC2c2-crRNA complex against on-target ssRNA and non-targetingcomplementary ssRNA. Bottom panels: electrophoretic mobility shift assaywith HEPN mutant R1278A LshC2c2-crRNA complex against on-target ssRNAand non-targeting complementary ssRNA.

FIGS. 112A-112D. Effect of RNA target-crRNA mismatches on LshC2c2 RNAseactivity. FIG. 112A. Quantitation of MS2 plaque assay testing singlemismatches at various positions in the spacer. Single mismatches haveminimal effect on phage interference. Locations of mismatches are shownin red; all mismatches are transversions. FIG. 112B. Quantitation of MS2plaque assay testing double mismatches at various positions in thespacer. Consecutive double mismatches in the middle of the spacereliminate phage interference. Locations of mismatches are shown in red;all mismatches are transversions. FIG. 112C. in vitro Lshc2c2 cleavageassaying single mismatches in the crRNA. Single mismatches have minimaleffect on ssRNA cleavage. Locations of mismatches are shown in red; allmismatches are transversions. FIG. 112D. in vitro Lshc2c2 cleavageassaying double mismatches in the crRNA. Consecutive double mismatchesin the middle of the crRNA abrogate LshC2c2 activity. Locations ofmismatches are shown in red; all mismatches are transversions

FIG. 113-1-113-3 Cleavage of three ssRNA targets that have the samecrRNA target sequence flanked by different sequences. The secondarystructure and sequence of each of the three targets is shown (top) andthe cleavage patterns of each target by C2c2 are shown using a 10% PAGEgel (bottom).

FIGS. 114A-114B-2. Mapping of C2c2 cleavage sites by RNA sequencing 114Aby position; 114B-1-B-2 by secondary structure. A Plots of the frequenceof cleavage ends observed in RNA-sequencing data for 5′ anchoredfragments. This data is projected onto secondary structure in (114B).FIG. 114B-1-B-2 The cleavage sites of targets 1 and 3 were mapped usingRNA-sequencing of the cleavage reaction. The frequency of ends of eachfragment (anchored at the 5′ end) are mapped to z-scores and projectedonto the secondary structure of the target.

FIGS. 115A-115I shows further target environment secondary structuresuseful for evaluation of C2c2 cleavage efficiency and cleavage sitemapping.

FIGS. 116A-116E. Heterologous expression of the Leptotrichia shahii C2c2locus mediates robust interference of RNA phage in Escherichia coli.FIG. 116A Schematic for the MS2 bacteriophage interference screen. Alibrary consisting of spacers targeting all possible sequences in theMS2 RNA genome was cloned into the LshC2c2 CRISPR array. Cellstransformed with the MS2-targeting spacer library were then treated withphage and plated, and surviving cells were harvested. The frequency ofspacers was compared to an untreated control (no phage), and enrichedspacers from the phage-treated condition were used for the generation ofPAM sequence logos. FIG. 116B Box plot showing the distribution ofnormalized crRNA frequencies for the phage-treated condition and controlscreen biological replicates (n=2). The box extends from the first tothird quartile with whiskers denoting the 1st and 99th percentiles. Themean is indicated by the red horizontal bar. FIG. 116C Sequence logogenerated from sequences flanking the 3′ end of protospacerscorresponding to enriched spacers, revealing the presence of a 3′ H PAM(not G). FIG. 116D Plaque assay used to validate the functionalsignificance of the H PAM in MS2 interference. All protospacers flankedby non-G PAMs exhibited robust phage interference. Spacer were designedto target the MS2 mat gene and their sequences are shown above theplaque images; the spacer used in the non-targeting control is notcomplementary to any sequence in either the E. coli or MS2 genome. Phagespots were applied as series of half-log dilutions. FIG. 116EQuantitation of MS2 plaque assay validating the H (non-G) PAMrequirement. 4 MS2-targeting spacers were designed for each PAM. Phagedilutions were spotted onto bacterial plates as series of half-logdilutions and interference was estimated based on the highest dilutionwithout plaques. Each point on the scatter plot represents the averageof three biological replicates and corresponds to a single spacer. Barsindicate the mean of 4 spacers for each PAM and errors are shown as thes.e.m.

FIGS. 117A-117D. LshC2c2 and crRNA mediate RNA-guided ssRNA cleavage.FIG. 117A Schematic of the ssRNA substrate being targeted by the crRNA.The protospacer region is highlighted in blue and the PAM is indicatedby the magenta bar. FIG. 117B A denaturing gel demonstratingcrRNA-mediated ssRNA cleavage by LshC2c2. The ssRNA target is either 5′labeled with IRDye 800 or 3′ labeled with Cy5. Cleavage requires thepresence of the crRNA and is abolished by addition of EDTA. Fourcleavage sites are observed. FIG. 117C A denaturing gel demonstratingthe requirement for an H PAM (not G). Four ssRNA substrates that areidentical except for the PAM base (indicated by the magenta X in theschematic) were used for the in vitro cleavage reactions. ssRNA cleavageactivity is dependent on the nucleotide immediately 3′ of the targetsite. FIG. 117D Schematic showing five protospacers for each PAM on thessRNA target (top). Denaturing gel showing crRNA-guided ssRNA cleavageactivity. crRNAs correspond to protospacer numbering.

FIGS. 118A-118I. C2c2 cleavage sites are determined by secondarystructure and sequence of the target RNA. FIG. 118A Schematic ofhomopolymer ssRNA targets. The protospacer is indicated by the lightblue bar. Homopolymer stretches of A (green) and U (red) bases areinterspaced by individual bases of G (orange) and C (purple). FIG. 118BDenaturing gel showing C2c2-crRNA-mediated cleavage patterns of eachhomopolymer. FIG. 118C Denaturing gel showing C2c2-crRNA-mediatedcleavage of three non-homopolymeric ssRNA targets (1, 4, 5) that sharethe same protospacer but are flanked by different sequences. Despiteidentical protospacers, different flanking sequences resulted indifferent cleavage patterns. (FIGS. 118D, 118F, and 118H) The cleavagesites of non-homopolymer ssRNA targets 1 FIG. 118D, 4 FIG. 118F, and 5FIG. 118H were mapped using RNA-sequencing of the cleavage products. Thefrequency of cleavage at each base is colored according to the z-scoreand shown on the predicted crRNA-ssRNA co-fold secondary structure.Fragments used to generate the frequency analysis contained the complete5′ end. The 5′ and 3′ end of the ssRNA target are indicated by blue andred outlines respectively. The 5′ and 3′ end of the spacer (outlined inyellow) is indicated by blue and orange highlights respectively. (118E,118G, and 118I) Plots of the frequencies of cleavage sites for eachposition of ssRNA targets 1, 4, and 5 for all reads that begin at the 5′end. The protospacer is indicated by the blue highlighted region.

FIGS. 119A-119E. The two HEPN domains of C2c2 are necessary forcrRNA-guided ssRNA cleavage but not for crRNA-guided ssRNA-binding. FIG.119A Schematic of the LshC2c2 locus and the domain organization of theLshC2c2 protein, showing conserved residues in HEPN domains. FIG. 119BQuantification of MS2 plaque assay with HEPN catalytic residue mutants.For each mutant, the same crRNA targeting protospacer 35 was used. FIG.119C Denaturing gel showing conserved residues of the HEPN motif arenecessary for crRNA-guided ssRNA cleavage. FIG. 119D Electrophoreticmobility shift assay (EMSA) evaluating affinity of the wild typeLshC2c2-crRNA complex against a targeted (left) and a non-targeted(right) ssRNA substrate. The non-targeted ssRNA substrate is thereverse-complement of the targeted ssRNA. EDTA is supplemented toreaction condition to reduce any cleavage activity. FIG. 119EElectrophoretic mobility shift assay with LshC2c2(R1278A)-crRNA complexagainst on-target ssRNA and non-targeting complementary ssRNA (samesubstrate sequences as in 119D).

FIGS. 120A-120B. RNA-guided RNase activity of LshC2c2 is dependent onspacer and direct repeat lengths. FIG. 120A Denaturing gel showingcrRNA-guided cleavage of ssRNA 1 as a function of spacer length. FIG.120B Denaturing gel showing crRNA-guided cleavage of ssRNA 1 as afunction of the direct repeat length.

FIGS. 121A-121B. RNA-guided RNase activity of LshC2c2 is dependent ondirect repeat structure and sequence. FIG. 121A Schematic showingmodifications to the crRNA direct repeat stem (top). Altered bases areshown in red. Denaturing gel showing crRNA-guided cleavage of ssRNA 1 byeach modified crRNA (bottom). FIG. 121B Schematic showing modificationsto the loop region of the crRNA direct repeat (top). Altered bases areshown in red and deletion lengths are indicated by arrows. Denaturinggel showing crRNA-guided cleavage of ssRNA 1 by each modified crRNA(bottom).

FIGS. 122A-122D. Effect of RNA target-crRNA mismatches on LshC2c2 RNaseactivity. FIG. 122A Quantification of MS2 plaque assays testing singlemismatches at various positions in the spacer. Single mismatches haveminimal effect on phage interference. Locations and identity ofmismatches are shown in red. FIG. 122B Quantification of MS2 plaqueassays testing double mismatches at various positions in the spacer.Consecutive double mismatches in the middle of the spacer eliminatephage interference. Locations and identity of mismatches are shown inred. FIG. 122C Schematic showing the position and identity of mismatches(red) in the crRNA spacer (top). Denaturing gel showing cleavage ofssRNA 1 guided by crRNAs with single mismatches in the spacer (bottom).FIG. 122D Schematic showing the position and identity of pairs ofmismatches (red) in the crRNA spacer (top). Denaturing gel showingcleavage of ssRNA 1 guided by crRNAs with pairs of mismatches in thespacer (bottom).

FIGS. 123A-123E. RFP mRNA knockdown by retargeting LshC2c2. FIG. 123ASchematic showing crRNA-guided knockdown of RFP in E. coliheterologously expressing the LshC2c2 locus. Three RFP-targeting spacerswere selected for each non-G PAM and each protospacer on the RFP mRNA isnumbered. FIG. 123B RFP mRNA-targeting spacers effected RFP knockdownwhereas DNA-targeting spacers (coding strand of the RFP gene on theexpression plasmid, indicated as “rc” spacers) did not affect RFPexpression. n=3 biological replicates. FIG. 123C Quantification of RFPknockdown in E. coli. Three spacers each targeting C, U, or APAM-flanking protospacers (9 spacers, numbered 5-13 as indicated inpanel (123A)) in the RFP mRNA were introduced and RFP expression wasmeasured by flow cytometry. Each point on the scatter plot representsthe average of three biological replicates and corresponds to a singlespacer. Bars indicate the mean of 4 spacers for each PAM and errors areshown as the s.e.m. FIG. 123D Timeline of E coli growth assay. FIG. 123EEffect of RFP mRNA targeting on the growth rate of E. coli transformedwith an inducible RFP expression plasmid as well as the LshC2c2 locuswith non-targeting, RNA targeting (spacer complementary to RFP genenon-coding strand), and DNA targeting (spacer complementary to RFP genecoding strand) spacers.

FIGS. 124A-124B. crRNA-guided target ssRNA cleavage activatesnon-specific RNase activity of LshC2c2. FIG. 124A Schematic of thebiochemical assay for assaying non-specific RNase activity onnon-crRNA-targeted collateral RNA molecules. In addition to theunlabeled crRNA-targeted ssRNA substrate, a second ssRNA with 3′fluorescent labeling is added to the same reaction to readoutnon-specific RNase activity. FIG. 124B Denaturing gel showingnon-specific RNase activity against non-targeted ssRNA substrates in thepresence of target RNA. The non-targeted ssRNA substrate is not cleavedin the absence of the crRNA-targeted ssRNA substate.

FIG. 125. C2c2 is an RNA adaptive immune system with possibleinvolvement in abortive infection via programmed cell death or dormancyinduction.

FIG. 126. RNA-sequencing of the Leptotrichia shahii locus heterologouslyexpressed in E. coli and spacer analysis. Adapted from S. Shmakov etal., Discovery and Functional Characterization of Diverse Class 2CRISPR-Cas Systems. Mol Cell 60, 385-397 (2015). Heterologous expressionof the LshC2c2 locus reveals processing of the array. Insert: In silicoco-folding analysis of a mature direct repeat.

FIGS. 127A-127D. MS2 phage screen replicates show agreement and do nothave a 5′ PAM. FIG. 127A Comparison of control (no phage) replicatesshow agreement and lack of enrichment or depletion. FIG. 127B Comparisonof phage replicates reveals both substantial depletion as well as anenriched population shared between both replicates. FIG. 127C Sequencelogo of 5′ sequence from enriched spacers admits no PAM. FIG. 127D Basefrequency of 5′ sequence from enriched spacers admits no 5′ PAM.

FIGS. 128A-128G. MS2 phage screen spacer representation across each PAM.FIG. 128A Box plot showing the distribution of spacer frequencies withspacers grouped by their 3′ PAM for phage treated conditions. Boxextends from the first to third quartile with the whiskers denoting the1st percentile and 99th percentile. ****, p<0.0001. FIG. 128B Multiplecomparison test (ANOVA with Tukey correction) between all possible PAMpairs for the phage treated spacer distributions. Plotted are theconfidence intervals for difference in means between the compared PAMpairs. FIG. 128C Box plot showing the distribution of spacer frequencieswith spacers grouped by their 3′ PAM for non-phage treated conditions.Box extends from the first to third quartile with the whiskers denotingthe 1st percentile and 99th percentile. ****, p<0.0001. FIG. 128DMultiple comparison test (ANOVA with Tukey correction) between allpossible PAM pairs for the non-phage treated spacer distributions.Plotted are the confidence intervals for difference in means between thecompared PAM pairs. FIG. 128E Cumulative frequency plots for the log 2normalized spacer counts. Spacers are separated by respective PAM toshow the enrichment differences between each PAM distribution. FIG. 128FThe average cumulative frequency difference between the phage and nophage cumulative frequency curves. The differences are shown for eachPAM distribution. FIG. 128G A zoomed in plot of the dotted box in (128F)to highlight the variation in enrichment between the different PAMs.

FIGS. 129A-129B. Top hits from MS2 phage screen show interference inplaque assay. FIG. 129A Images from validation of MS2 screen by plaqueassay showing reduced plaque formation in top hits. Phage dilutions werespotted on bacteria plates at decreasing numbers of plaque forming units(PFU). Spacer targets are shown above images; biological replicates arelabeled BR1, BR2, or BR3. Non-targeting control is the native LshC2c2locus. FIG. 129B Quantitation of MS2 plaque assay demonstratinginterference by top hits. Interference was quantified by highestdilution without plaques.

FIG. 130-1-130-2. MS2 plaque assay validates the 3′ H PAM. Four spacersfor each possible 3′ PAM (A, G, C, and U) are cloned into the pLshC2c2vector and tested for MS2 phage restriction in a plaque forming assay.The images show significantly reduced plaque formation for A, C, and UPAMs, and less restriction for the G PAM. Phage dilutions were spottedon bacteria plates at decreasing numbers of plaque forming units (PFU).Spacer targets are shown above images; three biological replicates arevertically stacked under each protospacer sequence. Non-targetingcontrols are the native LshC2c2 locus and the pACYC184 backbone.

FIGS. 131A-131D. Protein purification of LshC2c2. FIG. 131A Coomassieblue stained acrylamide gel of purified LshC2c2 stepwise purification. Astrong band just above 150 kD is consistent with the size of LshC2c2(171 kD). FIG. 131B Size exclusion gel filtration of LshC2c2. LshC2c2eluted at a size approximately >160 kD (62.9 mL). FIG. 131C Proteinstandards used to calibrate the Superdex 200 column. BDex=Blue Dextran(void volume), Ald=Aldolase (158 kD), Ov=Ovalbumin (44 kD),RibA=Ribonuclease A (13.7 kD), Apr=Aprotinin (6.5 kD). FIG. 131DCalibration curve of the Superdex 200 column. Kav is calculated as(elution volume−void volume)/(geometric column volume−void volume).Standards were plotted and fit to a logarithmic curve.

FIGS. 132A-132B. Further in vitro characterization of the RNA cleavagekinetics of LshC2c2. FIG. 132A A time series of LshC2c2 ssRNA cleavageusing a 5′- and 3-end-labeled target 1. FIG. 132B RNA-cleavage of 5′-and 3-end-labeled target 1 using LshC2c2-crRNA complex that is seriallydiluted in half-log steps.

FIG. 133. Characterization of the metal dependence of LshC2c2 RNAcleavage. A variety of divalent metal cations are supplemented for theLshC2c2 cleavage reaction using 5′-end-labeled target 1. Significantcleavage is only observed for Mg+2. Weak cleavage is observed for Ca+2and Mn+2.

FIGS. 134A-134D. LshC2c2 has no observable cleavage activity when usingdsRNA, dsDNA, or ssDNA substrates. FIG. 134A A schematic of the partialdsRNA target. 5′-end-labeled target 1 is annealed to two shorter RNAsthat are complementary to the regions flanking the protospacer site.This partial dsRNA is a more stringent test for dsRNA cutting since itshould still allow for LshC2c2 complex binding to ssRNA. FIG. 134BLshC2c2 cleavage activity of a dsRNA target shown in (134A) compared tothe ssRNA target 1. No cleavage is observed when using the dsRNAsubstrate. FIG. 134C LshC2c2 cleavage of a dsDNA plasmid library. Aplasmid library was generated to have seven randomized nucleotides 5′ ofprotospacer 14 to account for any sequence requirements for dsDNAcleavage. No cleavage is observed for this dsDNA library. FIG. 134D AssDNA version of target 1 is tested for cleavage by LshC2c2. No cleavageis observed.

FIGS. 135A-135B. LshC2c2 has no observable cleavage activity on dsDNAtargets in a co-transcriptional cleavage assay. FIG. 135A Schematic ofco-transcriptional cleavage assay. C2c2 was incubated with E. coli RNApolymerase (RNAP) elongation complexes and rNTP as previously described(P. Samai et al., Co-transcriptional DNA and RNA Cleavage during TypeIII CRISPR-Cas Immunity. Cell 161, 1164-1174 (2015)). FIG. 135B LshC2c2cleavage of DNA target after co-transcriptional cleavage assay. Nocleavage is observed.

FIG. 136. MS2 restriction assay reveals that single HEPN mutantsabrogate LshC2c2 activity. All four possible single HEPN mutants weregenerated in the pLshC2c2 vector (R597A, H602A, R1278A, and H1283A) withprotospacer 1. Images from plaque assay testing these HEPN mutant locishow similar plaque formation to the non-targeting locus and issignificantly higher than the WtC2c2 locus. Phage dilutions were spottedon bacteria plates at decreasing numbers of plaque forming units (PFU).Spacer targets are shown above images; biological replicates are labeledBR1, BR2, or BR3. Non-targeting control is the native LshC2c2 locus.

FIGS. 137A-137F. Quantitation of LshC2c2 binding. FIG. 137A Calculationof binding affinity for wildtype LshC2c2-crRNA complex and on-targetssRNA. Fraction of protein bound was quantified by densitometry fromFIG. 4D and KD was calculated by fitting to binding isotherm. FIG. 137BCalculation of binding affinity for HEPN mutant R1278A LshC2c2-crRNAcomplex and on-target ssRNA. Fraction of protein bound was quantified bydensitometry from FIG. 4E and KD was calculated by fitting to bindingisotherm. FIG. 137C Electrophoretic mobility shift assay with HEPNmutant R1278A LshC2c2 against on-target ssRNA in the absence of crRNA.EDTA is supplemented to reaction condition. FIG. 137D Electrophoreticmobility shift assay crRNA against on-target ssRNA. EDTA is supplementedto reaction condition. FIG. 137E Calculation of binding affinity forHEPN mutant R1278A LshC2c2 and on-target ssRNA in the absence of crRNA.Fraction of protein bound was quantified by densitometry from Fig. S12Cand KD was calculated by fitting to binding isotherm. FIG. 137FCalculation of binding affinity for crRNA and on-target ssRNA. Fractionof crRNA bound was quantified by densitometry from Fig. S12D and KD wascalculated by fitting to binding isotherm.

FIG. 138. MS2 restriction assay testing the effect of single and doublemismatches on LshC2c2 activity. pLshC2c2 with protospacer 41 wasmodified to have a series of single mismatches and consecutive doublemismatches as shown. Images from plaque assay testing these mismatchedspacers reveals reduced plaque formation for the single-mismatch spacerson-par with the fully complementary spacer. The double mismatch spacersshow increased plaque formation for a seed region in the middle ofspacer sequence. Phage dilutions were spotted on bacteria plates atdecreasing numbers of plaque forming units (PFU). Spacer targets areshown above images; biological replicates are labeled BR1, BR2, or BR3.Non-targeting control is the native LshC2c2 locus.

FIG. 139. HEPN mutant LshC2c2 are tested for RFP mRNA targetingactivity. The pLshC2c2 vector with protospacer 36 was modified to havethe single HEPN mutants R597A and R1278A (one in each of the HEPNdomains). These mutations resulted in little detectable RFP knockdown asmeasured by flow cytometry on the E coli.

FIGS. 140A-140B. Biochemical characterization of the collateral cleavageeffect. FIG. 140A LshCc2 is incubated with a crRNA targeting protospacer14 with and without unlabeled ssRNA target 1 (contains protospacer 14).When LshC2c2 is in the presence of target 1, significant cleavageactivity is observed for fluorescently labeled non-complementary targets6-9. FIG. 140B HEPN mutant collateral activity is compared to WT C2c2.The proteins are incubated with crRNA complementary to protospacer 14and with and without unlabeled homopolymer targets 2 or 3 (bothcontaining protospacer 14). The collateral effect is no longer observedwith the HEPN mutant proteins on the fluorescently labelednon-complementary target 8.

FIGS. 141A-141B. Growth suppression is correlated with RFP knockdown incells that express C2c2 and RFP-targeting crRNA. FIG. 141A shows theeffect on growth of inducible RFP expression in E. coli expressing C2c2and a crRNA that targets RFP. RFP was expressed using ananhydrotetracycline (aTc)-inducible gene expression system. Cellcultures were treated with aTc at the concentrations indicated.Increasing aTc expression suppressed growth. FIG. 141B shows the effecton growth of inducible RFP expression when a non-targeting crRNA wassubstituted for the RFP-targeting crRNA.

FIGS. 142A-142D. C2c2 preferentially cleaves at poly U tracts. ssRNAcleavage by C2c2 was examined using ssRNA substrate containing orlacking a poly U tract. End-labeled ssRNA was incubated with C2c2 andcrRNA and digestion products resolved. Poly U-containing substrate RNAwas efficiently cleaved. Substrate cleavage was crRNA-dependent as shownby absence of digestion products when C2c2 but not crRNA was present.FIGS. 142A and 142B: 3′-end labeled substrate. FIGS. 142C and 142D:5′-end labeled substrate. FIGS. 142 B and 142D indicate substratecleavage was efficient and specific as substrate concentration wasincreased.

FIGS. 143A-143B. Cleavage of target RNA with mismatched crRNA isposition dependent. ssRNA target was treated with crRNA containingdouble (FIG. 143A) or triple (FIG. 143B) mismatches and LshC2c2.Products of cleavage reactions were separated by electrophoresis.

FIG. 144. Cleavage of target RNA is sensitive to mutations and deletionsin the 3′ direct repeat region. ssRNA target was treated with LshC2c2and crRNA containing mismatches or deletions designed to disruptsecondary structure in the DR region. Products of cleavage reactionswere separated by electrophoresis.

FIGS. 145A-145B. C2c2 and MS2-crRNA can bind ssRNA. FIG. 145A. Bindingof a LshC2c2(R1278A) which is defective for substrate cleavage, to ssRNAwas tested. FIG. 145B: LshC2c2(R1278A) and MS2-crRNA were incubated withincreasing amounts of labeled ssRNA 10 (left panel) or labeled reversecomplement (RC) of ssRNA 10 (right panel). (See Table B for ssRNA 10sequence).

FIG. 146. C2c2 complex does not bind ssDNA. LshC2c2(R1278A) andMS2-crRNA were incubated with increasing amounts of ssDNA 10 (leftpanel) or ssDNA 10 reverse complement (RC) (right panel). (See Table Bfor ssRNA 10 sequence).

The figures herein are for illustrative purposes only and are notnecessarily drawn to scale.

DETAILED DESCRIPTION OF THE INVENTION

In general, a CRISPR-Cas or CRISPR system as used in the foregoingdocuments, such as WO 2014/093622 (PCT/US2013/074667) and referscollectively to transcripts and other elements involved in theexpression of or directing the activity of CRISPR-associated (“Cas”)genes, including sequences encoding a Cas gene, a tracr(trans-activating CRISPR) sequence (e.g. tracrRNA or an active partialtracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and atracrRNA-processed partial direct repeat in the context of an endogenousCRISPR system), a guide sequence (also referred to as a “spacer” in thecontext of an endogenous CRISPR system), or “RNA(s)” as that term isherein used (e.g., RNA(s) to guide Cas, such as Cas9, e.g. CRISPR RNAand transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimericRNA)) or other sequences and transcripts from a CRISPR locus. Ingeneral, a CRISPR system is characterized by elements that promote theformation of a CRISPR complex at the site of a target sequence (alsoreferred to as a protospacer in the context of an endogenous CRISPRsystem). When the CRISPR protein is a C2c2 protein, a tracrRNA is notrequired.

In the context of formation of a CRISPR complex, “target sequence”refers to a sequence to which a guide sequence is designed to havecomplementarity, where hybridization between a target sequence and aguide sequence promotes the formation of a CRISPR complex. A targetsequence may comprise any polynucleotide, such as DNA or RNApolynucleotides. In some embodiments, a target sequence is located inthe nucleus or cytoplasm of a cell. In some embodiments, direct repeatsmay be identified in silico by searching for repetitive motifs thatfulfill any or all of the following criteria: 1. found in a 2 Kb windowof genomic sequence flanking the type II CRISPR locus; 2. span from 20to 50 bp; and 3. interspaced by 20 to 50 bp. In some embodiments, 2 ofthese criteria may be used, for instance 1 and 2, 2 and 3, or 1 and 3.In some embodiments, all 3 criteria may be used.

In embodiments of the invention the terms guide sequence and guide RNA,i.e. RNA capable of guiding Cas to a target genomic locus, are usedinterchangeably as in foregoing cited documents such as WO 2014/093622(PCT/US2013/074667). In general, a guide sequence is any polynucleotidesequence having sufficient complementarity with a target polynucleotidesequence to hybridize with the target sequence and directsequence-specific binding of a CRISPR complex to the target sequence. Insome embodiments, the degree of complementarity between a guide sequenceand its corresponding target sequence, when optimally aligned using asuitable alignment algorithm, is about or more than about 50%, 60%, 75%,80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may bedetermined with the use of any suitable algorithm for aligningsequences, non-limiting example of which include the Smith-Watermanalgorithm, the Needleman-Wunsch algorithm, algorithms based on theBurrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW,Clustal X, BLAT, Novoalign (Novocraft Technologies; available atwww.novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (availableat soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). Insome embodiments, a guide sequence is about or more than about 5, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In someembodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30,25, 20, 15, 12, or fewer nucleotides in length. Preferably the guidesequence is 10 30 nucleotides long. The ability of a guide sequence todirect sequence-specific binding of a CRISPR complex to a targetsequence may be assessed by any suitable assay. For example, thecomponents of a CRISPR system sufficient to form a CRISPR complex,including the guide sequence to be tested, may be provided to a hostcell having the corresponding target sequence, such as by transfectionwith vectors encoding the components of the CRISPR sequence, followed byan assessment of preferential cleavage within the target sequence, suchas by Surveyor assay as described herein. Similarly, cleavage of atarget polynucleotide sequence may be evaluated in a test tube byproviding the target sequence, components of a CRISPR complex, includingthe guide sequence to be tested and a control guide sequence differentfrom the test guide sequence, and comparing binding or rate of cleavageat the target sequence between the test and control guide sequencereactions. Other assays are possible, and will occur to those skilled inthe art.

In a classic CRISPR-Cas systems, the degree of complementarity between aguide sequence and its corresponding target sequence can be about ormore than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%;a guide or RNA or sgRNA can be about or more than about 5, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,35, 40, 45, 50, 75, or more nucleotides in length; or guide or RNA orsgRNA can be less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, orfewer nucleotides in length. However, an aspect of the invention is toreduce off-target interactions, e.g., reduce the guide interacting witha target sequence having low complementarity. Indeed, in the examples,it is shown that the invention involves mutations that result in theCRISPR-Cas system being able to distinguish between target andoff-target sequences that have greater than 80% to about 95%complementarity, e.g., 83%-84% or 88-89% or 94-95% complementarity (forinstance, distinguishing between a target having 18 nucleotides from anoff-target of 18 nucleotides having 1, 2 or 3 mismatches). Accordingly,in the context of the present invention the degree of complementaritybetween a guide sequence and its corresponding target sequence isgreater than 94.5% or 95% or 95.5% or 96% or 96.5% or 97% or 97.5% or98% or 98.5% or 99% or 99.5% or 99.9%, or 100%. Off target is less than100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or96.5% or 96% or 95.5% or 95% or 94.5% or 94% or 93% or 92% or 91% or 90%or 89% or 88% or 87% or 86% or 85% or 84% or 83% or 82% or 81% or 80%complementarity between the sequence and the guide, with it advantageousthat off target is 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98%or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% complementaritybetween the sequence and the guide.

In certain embodiments, modulations of cleavage efficiency can beexploited by introduction of mismatches, e.g. 1 or more mismatches, suchas 1 or 2 mismatches between spacer sequence and target sequence,including the position of the mismatch along the spacer/target. The morecentral (i.e. not 3′ or 5′) for instance a double mismatch is, the morecleavage efficiency is affected. Accordingly, by chosing mismatchposition along the spacer, cleavage efficiency can be modulated. Bymeans of example, if less than 100% cleavage of targets is desired (e.g.in a cell population), 1 or more, such as preferably 2 mismatchesbetween spacer and target sequence may be introduced in the spacersequences. The more central along the spacer of the mismatch position,the lower the cleavage percentage.

The methods according to the invention as described herein comprehendinducing one or more nucleotide modifications in a eukaryotic cell (invitro, i.e. in an isolated eukaryotic cell) as herein discussedcomprising delivering to cell a vector as herein discussed. Themutation(s) can include the introduction, deletion, or substitution ofone or more nucleotides at each target sequence of cell(s) via theguide(s) RNA(s) or sgRNA(s). The mutations can include the introduction,deletion, or substitution of 1-75 nucleotides at each target sequence ofsaid cell(s) via the guide(s) RNA(s). The mutations can include theintroduction, deletion, or substitution of 1, 5, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45,50, or 75 nucleotides at each target sequence of said cell(s) via theguide(s) RNA(s). The mutations can include the introduction, deletion,or substitution of 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides ateach target sequence of said cell(s) via the guide(s) RNA(s). Themutations include the introduction, deletion, or substitution of 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 35, 40, 45, 50, or 75 nucleotides at each target sequence of saidcell(s) via the guide(s) RNA(s). The mutations can include theintroduction, deletion, or substitution of 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at each targetsequence of said cell(s) via the guide(s) RNA(s). The mutations caninclude the introduction, deletion, or substitution of 40, 45, 50, 75,100, 200, 300, 400 or 500 nucleotides at each target sequence of saidcell(s) via the guide(s) RNA(s).

For minimization of toxicity and off-target effect, it will be importantto control the concentration of Cas mRNA or protein and guide RNAdelivered. Optimal concentrations of Cas mRNA or protein and guide RNAcan be determined by testing different concentrations in a cellular ornon-human eukaryote animal model and using deep sequencing the analyzethe extent of modification at potential off-target genomic loci.

Typically, in the context of an endogenous CRISPR system, formation of aCRISPR complex (comprising a guide sequence hybridized to a targetsequence and complexed with one or more Cas proteins) results incleavage in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50,or more base pairs from) the target sequence.

The nucleic acid molecule encoding a Cas is advantageously codonoptimized Cas. An example of a codon optimized sequence, is in thisinstance a sequence optimized for expression in a eukaryote, e.g.,humans (i.e. being optimized for expression in humans), or for anothereukaryote, animal or mammal as herein discussed; see, e.g., SaCas9 humancodon optimized sequence in WO 2014/093622 (PCT/US2013/074667). Whilstthis is preferred, it will be appreciated that other examples arepossible and codon optimization for a host species other than human, orfor codon optimization for specific organs is known. In someembodiments, an enzyme coding sequence encoding a Cas is codon optimizedfor expression in particular cells, such as eukaryotic cells. Theeukaryotic cells may be those of or derived from a particular organism,such as a mammal, including but not limited to human, or non-humaneukaryote or animal or mammal as herein discussed, e.g., mouse, rat,rabbit, dog, livestock, or non-human mammal or primate. In someembodiments, processes for modifying the germ line genetic identity ofhuman beings and/or processes for modifying the genetic identity ofanimals which are likely to cause them suffering without any substantialmedical benefit to man or animal, and also animals resulting from suchprocesses, may be excluded. In general, codon optimization refers to aprocess of modifying a nucleic acid sequence for enhanced expression inthe host cells of interest by replacing at least one codon (e.g. aboutor more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) ofthe native sequence with codons that are more frequently or mostfrequently used in the genes of that host cell while maintaining thenative amino acid sequence. Various species exhibit particular bias forcertain codons of a particular amino acid. Codon bias (differences incodon usage between organisms) often correlates with the efficiency oftranslation of messenger RNA (mRNA), which is in turn believed to bedependent on, among other things, the properties of the codons beingtranslated and the availability of particular transfer RNA (tRNA)molecules. The predominance of selected tRNAs in a cell is generally areflection of the codons used most frequently in peptide synthesis.Accordingly, genes can be tailored for optimal gene expression in agiven organism based on codon optimization. Codon usage tables arereadily available, for example, at the “Codon Usage Database” availableat www.kazusa.orjp/codon/ and these tables can be adapted in a number ofways. See Nakamura, Y., et al. “Codon usage tabulated from theinternational DNA sequence databases: status for the year 2000” Nucl.Acids Res. 28:292 (2000). Computer algorithms for codon optimizing aparticular sequence for expression in a particular host cell are alsoavailable, such as Gene Forge (Aptagen; Jacobus, Pa.), are alsoavailable. In some embodiments, one or more codons (e.g. 1, 2, 3, 4, 5,10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a Cascorrespond to the most frequently used codon for a particular aminoacid.

In certain embodiments, the methods as described herein may compriseproviding a Cas transgenic cell in which one or more nucleic acidsencoding one or more guide RNAs are provided or introduced operablyconnected in the cell with a regulatory element comprising a promoter ofone or more gene of interest. As used herein, the term “Cas transgeniccell” refers to a cell, such as a eukaryotic cell, in which a Cas genehas been genomically integrated. The nature, type, or origin of the cellare not particularly limiting according to the present invention. Alsothe way how the Cas transgene is introduced in the cell is may vary andcan be any method as is known in the art. In certain embodiments, theCas transgenic cell is obtained by introducing the Cas transgene in anisolated cell. In certain other embodiments, the Cas transgenic cell isobtained by isolating cells from a Cas transgenic organism. By means ofexample, and without limitation, the Cas transgenic cell as referred toherein may be derived from a Cas transgenic eukaryote, such as a Casknock-in eukaryote. Reference is made to WO 2014/093622(PCT/US13/74667), incorporated herein by reference. Methods of US PatentPublication Nos. 20120017290 and 20110265198 assigned to SangamoBioSciences, Inc. directed to targeting the Rosa locus may be modifiedto utilize the CRISPR Cas system of the present invention. Methods of USPatent Publication No. 20130236946 assigned to Cellectis directed totargeting the Rosa locus may also be modified to utilize the CRISPR Cassystem of the present invention. By means of further example referenceis made to Platt et. al. (Cell; 159(2):440-455 (2014)), describing aCas9 knock-in mouse, which is incorporated herein by reference. The Castransgene can further comprise a Lox-Stop-polyA-Lox(LSL) cassettethereby rendering Cas expression inducible by Cre recombinase.Alternatively, the Cas transgenic cell may be obtained by introducingthe Cas transgene in an isolated cell. Delivery systems for transgenesare well known in the art. By means of example, the Cas transgene may bedelivered in for instance eukaryotic cell by means of vector (e.g., AAV,adenovirus, lentivirus) and/or particle and/or nanoparticle delivery, asalso described herein elsewhere.

It will be understood by the skilled person that the cell, such as theCas transgenic cell, as referred to herein may comprise further genomicalterations besides having an integrated Cas gene or the mutationsarising from the sequence specific action of Cas when complexed with RNAcapable of guiding Cas to a target locus, such as for instance one ormore oncogenic mutations, as for instance and without limitationdescribed in Platt et al. (2014), Chen et al., (2014) or Kumar et al.(2009).

In some embodiments, the Cas sequence is fused to one or more nuclearlocalization sequences (NLSs), such as about or more than about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the Cascomprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore NLSs at or near the amino-terminus, about or more than about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy-terminus,or a combination of these (e.g. zero or at least one or more NLS at theamino-terminus and zero or at one or more NLS at the carboxy terminus).When more than one NLS is present, each may be selected independently ofthe others, such that a single NLS may be present in more than one copyand/or in combination with one or more other NLSs present in one or morecopies. In a preferred embodiment of the invention, the Cas comprises atmost 6 NLSs. In some embodiments, an NLS is considered near the N- orC-terminus when the nearest amino acid of the NLS is within about 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along thepolypeptide chain from the N- or C-terminus. Non-limiting examples ofNLSs include an NLS sequence derived from: the NLS of the SV40 viruslarge T-antigen, having the amino acid sequence PKKKRKV(SEQ ID NO: X);the NLS from nucleoplasmin (e.g. the nucleoplasmin bipartite NLS withthe sequence KRPAATKKAGQAKKKK) (SEQ ID NO: X); the c-myc NLS having theamino acid sequence PAAKRVKLD (SEQ ID NO: X) or RQRRNELKRSP(SEQ ID NO:X); the hRNPA1 M9 NLS having the sequenceNQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY(SEQ ID NO: X); the sequenceRMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: X) of the IBBdomain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: X) andPPKKARED (SEQ ID NO: X) of the myoma T protein; the sequence POPKKKPL(SEQ ID NO: X) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: X) ofmouse c-abl IV; the sequences DRLRR (SEQ ID NO: X) and PKQKKRK (SEQ IDNO: X) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO:X) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ IDNO: X) of the mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQID NO: X) of the human poly(ADP-ribose) polymerase; and the sequenceRKCLQAGMNLEARKTKK (SEQ ID NO: X) of the steroid hormone receptors(human) glucocorticoid. In general, the one or more NLSs are ofsufficient strength to drive accumulation of the Cas in a detectableamount in the nucleus of a eukaryotic cell. In general, strength ofnuclear localization activity may derive from the number of NLSs in theCas, the particular NLS(s) used, or a combination of these factors.Detection of accumulation in the nucleus may be performed by anysuitable technique. For example, a detectable marker may be fused to theCas, such that location within a cell may be visualized, such as incombination with a means for detecting the location of the nucleus (e.g.a stain specific for the nucleus such as DAPI). Cell nuclei may also beisolated from cells, the contents of which may then be analyzed by anysuitable process for detecting protein, such as immunohistochemistry,Western blot, or enzyme activity assay. Accumulation in the nucleus mayalso be determined indirectly, such as by an assay for the effect ofCRISPR complex formation (e.g. assay for DNA cleavage or mutation at thetarget sequence, or assay for altered gene expression activity affectedby CRISPR complex formation and/or Cas enzyme activity), as compared toa control no exposed to the Cas or complex, or exposed to a Cas lackingthe one or more NLSs or NESs. In certain embodiments, other localizationtags may be fused to the Cas protein, such as without limitation forlocalizing the Cas to particular sites in a cell, such as organells,such mitochondria, plastids, chloroplast, vesicles, golgi, (nuclear orcellular) membranes, ribosomes, nucleoluse, ER, cytoskeleton, vacuoles,centrosome, nucleosome, granules, centrioles, etc.

In certain aspects the invention involves vectors, e.g. for deliveringor introducing in a cell Cas and/or RNA capable of guiding Cas to atarget locus (i.e. guide RNA), but also for propagating these components(e.g. in prokaryotic cells). A used herein, a “vector” is a tool thatallows or facilitates the transfer of an entity from one environment toanother. It is a replicon, such as a plasmid, phage, or cosmid, intowhich another DNA segment may be inserted so as to bring about thereplication of the inserted segment. Generally, a vector is capable ofreplication when associated with the proper control elements. Ingeneral, the term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. Vectorsinclude, but are not limited to, nucleic acid molecules that aresingle-stranded, double-stranded, or partially double-stranded; nucleicacid molecules that comprise one or more free ends, no free ends (e.g.circular); nucleic acid molecules that comprise DNA, RNA, or both; andother varieties of polynucleotides known in the art. One type of vectoris a “plasmid,” which refers to a circular double stranded DNA loop intowhich additional DNA segments can be inserted, such as by standardmolecular cloning techniques. Another type of vector is a viral vector,wherein virally-derived DNA or RNA sequences are present in the vectorfor packaging into a virus (e.g. retroviruses, replication defectiveretroviruses, adenoviruses, replication defective adenoviruses, andadeno-associated viruses (AAVs)). Viral vectors also includepolynucleotides carried by a virus for transfection into a host cell.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g. bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively-linked. Such vectors are referred to herein as “expressionvectors.” Common expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell). With regards torecombination and cloning methods, mention is made of U.S. patentapplication Ser. No. 10/815,730, published Sep. 2, 2004 as US2004-0171156 A1, the contents of which are herein incorporated byreference in their entirety.

The vector(s) can include the regulatory element(s), e.g., promoter(s).The vector(s) can comprise Cas encoding sequences, and/or a single, butpossibly also can comprise at least 3 or 8 or 16 or 32 or 48 or 50 guideRNA(s) (e.g., sgRNAs) encoding sequences, such as 1-2, 1-3, 1-4 1-5,3-6, 3-7, 3-8, 3-9, 3-10, 3-8, 3-16, 3-30, 3-32, 3-48, 3-50 RNA(s)(e.g., sgRNAs). In a single vector there can be a promoter for each RNA(e.g., sgRNA), advantageously when there are up to about 16 RNA(s); and,when a single vector provides for more than 16 RNA(s), one or morepromoter(s) can drive expression of more than one of the RNA(s), e.g.,when there are 32 RNA(s), each promoter can drive expression of twoRNA(s), and when there are 48 RNA(s), each promoter can drive expressionof three RNA(s). By simple arithmetic and well established cloningprotocols and the teachings in this disclosure one skilled in the artcan readily practice the invention as to the RNA(s) for a suitableexemplary vector such as AAV, and a suitable promoter such as the U6promoter. For example, the packaging limit of AAV is ˜4.7 kb. The lengthof a single U6-gRNA (plus restriction sites for cloning) is 361 bp.Therefore, the skilled person can readily fit about 12-16, e.g., 13U6-gRNA cassettes in a single vector. This can be assembled by anysuitable means, such as a golden gate strategy used for TALE assembly(http://www.genome-engineering.org/taleffectors/). The skilled personcan also use a tandem guide strategy to increase the number of U6-gRNAsby approximately 1.5 times, e.g., to increase from 12-16, e.g., 13 toapproximately 18-24, e.g., about 19 U6-gRNAs. Therefore, one skilled inthe art can readily reach approximately 18-24, e.g., about 19promoter-RNAs, e.g., U6-gRNAs in a single vector, e.g., an AAV vector. Afurther means for increasing the number of promoters and RNAs in avector is to use a single promoter (e.g., U6) to express an array ofRNAs separated by cleavable sequences. And an even further means forincreasing the number of promoter-RNAs in a vector, is to express anarray of promoter-RNAs separated by cleavable sequences in the intron ofa coding sequence or gene; and, in this instance it is advantageous touse a polymerase II promoter, which can have increased expression andenable the transcription of long RNA in a tissue specific manner. (see,e.g., http://nar.oxfordjournals.org/content/34/7/e53. short,http://www.nature.com/mt/journal/v16/n9/abs/mt2008144a.html). In anadvantageous embodiment, AAV may package U6 tandem gRNA targeting up toabout 50 genes. Accordingly, from the knowledge in the art and theteachings in this disclosure the skilled person can readily make and usevector(s), e.g., a single vector, expressing multiple RNAs or guidesunder the control or operatively or functionally linked to one or morepromoters-especially as to the numbers of RNAs or guides discussedherein, without any undue experimentation.

The guide RNA(s) encoding sequences and/or Cas encoding sequences, canbe functionally or operatively linked to regulatory element(s) and hencethe regulatory element(s) drive expression. The promoter(s) can beconstitutive promoter(s) and/or conditional promoter(s) and/or induciblepromoter(s) and/or tissue specific promoter(s). The promoter can beselected from the group consisting of RNA polymerases, pol I, pol II,pol III, T7, U6, H1, retroviral Rous sarcoma virus (RSV) LTR promoter,the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolatereductase promoter, the β-actin promoter, the phosphoglycerol kinase(PGK) promoter, and the EF1α promoter. An advantageous promoter is thepromoter is U6.

The CRISPR-Cas loci has more than 50 gene families and there is nostrictly universal genes. Therefore, no single evolutionary tree isfeasible and a multi-pronged approach is needed to identify newfamilies. So far, there is comprehensive cas gene identification of 395profiles for 93 Cas proteins. Classification includes signature geneprofiles plus signatures of locus architecture. A new classification ofCRISPR-Cas systems is proposed in FIGS. 1A and 1B. Class 1 includesmultisubunit crRNA-effector complexes (Cascade) and Class 2 includesSingle-subunit crRNA-effector complexes (Cas9-like). FIG. 2 provides amolecular organization of CRISPR-Cas. FIG. 3 provides structures of TypeI and III effector complexes: common architecture/common ancestrydespite extensive sequence divergence. FIG. 4 shows CRISPR-Cas as a RNArecognition motif (RRM)-centered system. FIG. 5 shows Cas1 phylogenywhere recombination of adaptation and crRNA-effector modules show amajor aspect of CRISPR-Cas evolution. FIG. 6 shows a CRISPR-Cas census,specifically a distribution of CRISPR-Cas types/subtypes among archaeaand bacteria.

The action of the CRISPR-Cas system is usually divided into threestages: (1) adaptation or spacer integration, (2) processing of theprimary transcript of the CRISPR locus (pre-crRNA) and maturation of thecrRNA which includes the spacer and variable regions corresponding to 5′and 3′ fragments of CRISPR repeats, and (3) DNA or RNA interference. Twoproteins, Cas1 and Cas2, that are present in the great majority of theknown CRISPR-Cas systems are sufficient for the insertion of spacersinto the CRISPR cassettes. These two proteins form a complex that isrequired for this adaptation process; the endonuclease activity of Cas1is required for spacer integration whereas Cas2 appears to perform anonenzymatic function. The Cas1-Cas2 complex represents the highlyconserved “information processing” module of CRISPR-Cas that appears tobe quasi-autonomous from the rest of the system. (See Annotation andClassification of CRISPR-Cas Systems. Makarova K S, Koonin E V. MethodsMol Biol. 2015; 1311:47-75).

The previously described Class 2 systems, namely Type II and theputative Type V, consisted of only three or four genes in the casoperon, namely the cas1 and cas2 genes comprising the adaptation module(the cas1-cas2 pair of genes are not involved in interference), a singlemultidomain effector protein that is responsible for interference butalso contributes to the pre-crRNA processing and adaptation, and often afourth gene with uncharacterized functions that is dispensable in atleast some Type II systems (and in some cases the fourth gene is cas4(biochemical or in silico evidence shows that Cas4 is a PD-(DE)xKsuperfamily nuclease with three-cysteine C-terminal cluster; possesses5′-ssDNA exonuclease activity) or csn2, which encodes an inactivatedATPase). In most cases, a CRISPR array and a gene for a distinct RNAspecies known as tracrRNA, a trans-encoded small CRISPR RNA, areadjacent to Class 2 cas operons. The tracrRNA is partially homologous tothe repeats within the respective CRISPR array and is essential for theprocessing of pre-crRNA that is catalyzed by RNAse III, a ubiquitousbacterial enzyme that is not associated with the CRISPR-cas loci.

Cas1 is the most conserved protein that is present in most of theCRISPR-Cas systems and evolves slower than other Cas proteins.Accordingly, Cas1 phylogeny has been used as the guide for CRISPR-Cassystem classification. Biochemical or in silico evidence shows that Cas1is a metal-dependent deoxyribonuclease. Deletion of Cas1 in E. coliresults in increased sensitivity to DNA damage and impaired chromosomalsegregation as described in “A dual function of the CRISPR-Cassystem inbacterial antivirus immunity and DNA repair,” Babu M et al. MolMicrobiol 79:484-502 (2011). Biochemical or in silico evidence showsthat Cas 2 is a RNase specific to U-rich regions and is adouble-stranded DNase.

Aspects of the invention relate to the identification and engineering ofnovel effector proteins associated with Class 2 CRISPR-Cas systems. In apreferred embodiment, the effector protein comprises a single-subuniteffector module. In a further embodiment the effector protein isfunctional in prokaryotic or eukaryotic cells for in vitro, in vivo orex vivo applications. An aspect of the invention encompassescomputational methods and algorithms to predict new Class 2 CRISPR-Cassystems and identify the components therein.

In one embodiment, a computational method of identifying novel Class 2CRISPR-Cas loci comprises the following steps: detecting all contigsencoding the Cas1 protein; identifying all predicted protein codinggenes within 20 kB of the cas1 gene, more particularly within the region20 kb from the start of the cas1 gene and 20 kb from the end of the cas1gene; comparing the identified genes with Cas protein-specific profilesand predicting CRISPR arrays; selecting partial and/or unclassifiedcandidate CRISPR-Cas loci containing proteins larger than 500 aminoacids (>500 aa); analyzing selected candidates using PSI-BLAST andHHPred, thereby isolating and identifying novel Class 2 CRISPR-Cas loci.In addition to the above mentioned steps, additional analysis of thecandidates may be conducted by searching metagenomics databases foradditional homologs.

In one aspect the detecting all contigs encoding the Cas1 protein isperformed by GenemarkS which a gene prediction program as furtherdescribed in “GeneMarkS: a self-training method for prediction of genestarts in microbial genomes. Implications for finding sequence motifs inregulatory regions.” John Besemer, Alexandre Lomsadze and MarkBorodovsky, Nucleic Acids Research (2001) 29, pp 2607-2618, hereinincorporated by reference.

In one aspect the identifying all predicted protein coding genes iscarried out by comparing the identified genes with Cas protein-specificprofiles and annotating them according to NCBI Conserved Domain Database(CDD) which is a protein annotation resource that consists of acollection of well-annotated multiple sequence alignment models forancient domains and full-length proteins. These are available asposition-specific score matrices (PSSMs) for fast identification ofconserved domains in protein sequences via RPS-BLAST. CDD contentincludes NCBI-curated domains, which use 3D-structure information toexplicitly define domain boundaries and provide insights intosequence/structure/function relationships, as well as domain modelsimported from a number of external source databases (Pfam, SMART, COG,PRK, TIGRFAM). In a further aspect, CRISPR arrays were predicted using aPILER-CR program which is a public domain software for finding CRISPRrepeats as described in “PILER-CR: fast and accurate identification ofCRISPR repeats”, Edgar, R. C., BMC Bioinformatics, January 20;8:18(2007), herein incorporated by reference.

In a further aspect, the case by case analysis is performed usingPSI-BLAST (Position-Specific Iterative Basic Local Alignment SearchTool). PSI-BLAST derives a position-specific scoring matrix (PSSM) orprofile from the multiple sequence alignment of sequences detected abovea given score threshold using protein-protein BLAST. This PSSM is usedto further search the database for new matches, and is updated forsubsequent iterations with these newly detected sequences. Thus,PSI-BLAST provides a means of detecting distant relationships betweenproteins.

In another aspect, the case by case analysis is performed using HHpred,a method for sequence database searching and structure prediction thatis as easy to use as BLAST or PSI-BLAST and that is at the same timemuch more sensitive in finding remote homologs. In fact, HHpred'ssensitivity is competitive with the most powerful servers for structureprediction currently available. HHpred is the first server that is basedon the pairwise comparison of profile hidden Markov models (HMMs).Whereas most conventional sequence search methods search sequencedatabases such as UniProt or the NR, HHpred searches alignmentdatabases, like Pfam or SMART. This greatly simplifies the list of hitsto a number of sequence families instead of a clutter of singlesequences. All major publicly available profile and alignment databasesare available through HHpred. HHpred accepts a single query sequence ora multiple alignment as input. Within only a few minutes it returns thesearch results in an easy-to-read format similar to that of PSI-BLAST.Search options include local or global alignment and scoring secondarystructure similarity. HHpred can produce pairwise query-templatesequence alignments, merged query-template multiple alignments (e.g. fortransitive searches), as well as 3D structural models calculated by theMODELLER software from HHpred alignments.

The term “nucleic acid-targeting system”, wherein nucleic acid is DNA orRNA, and in some aspects may also refer to DNA-RNA hybrids orderivatives thereof, refers collectively to transcripts and otherelements involved in the expression of or directing the activity of DNAor RNA-targeting CRISPR-associated (“Cas”) genes, which may includesequences encoding a DNA or RNA-targeting Cas protein and a DNA orRNA-targeting guide RNA comprising a CRISPR RNA (crRNA) sequence and (insome but not all systems) a trans-activating CRISPR/Cas system RNA(tracrRNA) sequence, or other sequences and transcripts from a DNA orRNA-targeting CRISPR locus. In general, a RNA-targeting system ischaracterized by elements that promote the formation of a DNA orRNA-targeting complex at the site of a target DNA or RNA sequence. Inthe context of formation of a DNA or RNA-targeting complex, “targetsequence” refers to a DNA or RNA sequence to which a DNA orRNA-targeting guide RNA is designed to have complementarity, wherehybridization between a target sequence and a RNA-targeting guide RNApromotes the formation of a RNA-targeting complex. In some embodiments,a target sequence is located in the nucleus or cytoplasm of a cell.

In an aspect of the invention, novel RNA targeting systems also referredto as RNA- or RNA-targeting CRISPR/Cas or the CRISPR-Cas systemRNA-targeting system of the present application are based on identifiedType VI Cas proteins which do not require the generation of customizedproteins to target specific RNA sequences but rather a single enzyme canbe programmed by a RNA molecule to recognize a specific RNA target, inother words the enzyme can be recruited to a specific RNA target usingsaid RNA molecule.

In an aspect of the invention, novel DNA targeting systems also referredto as DNA- or DNA-targeting CRISPR/Cas or the CRISPR-Cas systemRNA-targeting system of the present application are based on identifiedType VI Cas proteins which do not require the generation of customizedproteins to target specific RNA sequences but rather a single enzyme canbe programmed by a RNA molecule to recognize a specific DNA target, inother words the enzyme can be recruited to a specific DNA target usingsaid RNA molecule.

The nucleic acids-targeting systems, the vector systems, the vectors andthe compositions described herein may be used in various nucleicacids-targeting applications, altering or modifying synthesis of a geneproduct, such as a protein, nucleic acids cleavage, nucleic acidsediting, nucleic acids splicing; trafficking of target nucleic acids,tracing of target nucleic acids, isolation of target nucleic acids,visualization of target nucleic acids, etc.

As used herein, a Cas protein or a CRISPR enzyme refers to any of theproteins presented in the new classification of CRISPR-Cas systems.

In an advantageous embodiment, the present invention encompasseseffector proteins identified in a Type VI CRISPR-Cas loci, e.g. the C2c2loci. Herein, C2c2 refers to Class 2 candidate 2. The C2c2 lociencompass cas1 and cas2 genes along with the large protein Applicantsdenote as C2c2p, and a CRISPR array; however, C2c2p is often encodednext to a CRISPR array but not cas1-cas2 (compare FIG. 9 and FIG. 15).

C2c2 Nuclease

The activity of C2c2 depends on the presence of two HEPN domains. Thesehave been shown to be RNase domains, i.e. nuclease (in particular anendonuclease) cutting RNA. C2c2 HEPN may also target DNA, or potentiallyDNA and/or RNA. On the basis that that the HEPN domains of C2c2 are atleast capable of binding to and, in their wild-type form, cutting RNA,then it is preferred that the C2c2 effector protein has RNase function.It may also, or alternatively, have DNase function.

Thus, in some embodiments, the effector protein may be a RNA-bindingprotein, such as a dead-Cas type effector protein, which may beoptionally functionalized as described herein for instance with antranscriptional activator or repressor domain, NLS or other functionaldomain. In some embodiments, the effector protein may be a RNA-bindingprotein that cleaves a single strand of RNA. If the RNA bound is ssRNA,then the ssRNA is fully cleaved. In some embodiments, the effectorprotein may be a RNA-binding protein that cleaves a double strand ofRNA, for example if it comprises two RNase domains. If the RNA bound isdsRNA, then the dsRNA is fully cleaved.

RNase function in CRISPR systems is known, for example mRNA targetinghas been reported for certain type III CRISPR-Cas systems (Hale et al.,2014, Genes Dev, vol. 28, 2432-2443; Hale et al., 2009, Cell, vol. 139,945-956; Peng et al., 2015, Nucleic acids research, vol. 43, 406-417)and provides significant advantages. In the Staphylococcus epidermistype III-A system, transcription across targets results in cleavage ofthe target DNA and its transcripts, mediated by independent active siteswithin the Cas10-Csm ribonucleoprotein effector complex (see, Samai etal., 2015, Cell, vol. 151, 1164-1174). A CRISPR-Cas system, compositionor method targeting RNA via the present effector proteins is thusprovided.

The target RNA, i.e. the RNA of interest, is the RNA to be targeted bythe present invention leading to the recruitment to, and the binding ofthe effector protein at, the target site of interest on the target RNA.The target RNA may be any suitable form of RNA. This may include, insome embodiments, mRNA. In other embodiments, the target RNA may includetRNA or rRNA. In other embodiments, the target RNA may include miRNA. Inother embodiments, the target RNA may include siRNA.

Interfering RNA (RNAi) and microRNA (miRNA)

In other embodiments, the target RNA may include interfering RNA, i.e.RNA involved in an RNA interference pathway, such as shRNA, siRNA and soforth. In other embodiments, the target RNA may include microRNA(miRNA). Control over interfering RNA or miRNA may help reduceoff-target effects (OTE) seen with those approaches by reducing thelongevity of the interfering RNA or miRNA in vivo or in vitro.

In certain embodiments, the target is not the miRNA itself, but themiRNA binding site of a miRNA target.

In certain embodiments, miRNAs may be sequestered (such as includingsubcellularly relocated). In certain embodiments, miRNAs may be cut,such as without limitation at hairpins.

In certain embodiments, miRNA processing (such as including turnover) isincreased or decreased.

If the effector protein and suitable guide are selectively expressed(for example spatially or temporally under the control of a suitablepromoter, for example a tissue- or cell cycle-specific promoter and/orenhancer) then this could be used to ‘protect’ the cells or systems (invivo or in vitro) from RNAi in those cells. This may be useful inneighbouring tissues or cells where RNAi is not required or for thepurposes of comparison of the cells or tissues where the effectorprotein and suitable guide are and are not expressed (i.e. where theRNAi is not controlled and where it is, respectively). The effectorprotein may be used to control or bind to molecules comprising orconsisting of RNA, such as ribozymes, ribosomes or riboswitches. Inembodiments of the invention, the RNA guide can recruit the effectorprotein to these molecules so that the effector protein is able to bindto them.

The protein system of the invention can be applied in areas of RNAitechnologies, without undue experimentation, from this disclosure,including therapeutic, assay and other applications (see, e.g., Guidi etal., PLoS Negl Trop Dis 9(5): e0003801. doi:10.1371/journal.pntd; Crottyet al., In vivo RNAi screens: concepts and applications. Shane Crotty .. . 2015 Elsevier Ltd. Published by Elsevier Inc., PesticideBiochemistry and Physiology (Impact Factor: 2.01). January 2015; 120.DOI: 10.1016/j.pestbp.2015.01.002 and Makkonen et al., Viruses 2015,7(4), 2099-2125; doi:10.3390/v7042099), because the present applicationprovides the foundation for informed engineering of the system.

Ribosomal RNA (rRNA)

For example, azalide antibiotics such as azithromycin, are well known.They target and disrupt the 50S ribosomal subunit. The present effectorprotein, together with a suitable guide RNA to target the 50S ribosomalsubunit, may be, in some embodiments, recruited to and bind to the 50Sribosomal subunit. Thus, the present effector protein in concert with asuitable guide directed at a ribosomal (especially the 50s ribosomalsubunit) target is provided. Use of this use effector protein in concertwith the suitable guide directed at the ribosomal (especially the 50sribosomal subunit) target may include antibiotic use. In particular, theantibiotic use is analogous to the action of azalide antibiotics, suchas azithromycin. In some embodiments, prokaryotic ribosomal subunits,such as the 70S subunit in prokaryotes, the 50S subunit mentioned above,the 30S subunit, as well as the 16S and 5S subunits may be targeted. Inother embodiments, eukaryotic ribosomal subunits, such as the 80Ssubunit in eukaryotes, the 60S subunit, the 40S subunit, as well as the28S, 18S. 5.8S and 5S subunits may be targeted.

In some embodiments, the effector protein may be a RNA-binding protein,optionally functionalized, as described herein. In some embodiments, theeffector protein may be a RNA-binding protein that cleaves a singlestrand of RNA. In either case, but particularly where the RNA-bindingprotein cleaves a single strand of RNA, then ribosomal function may bemodulated and, in particular, reduced or destroyed. This may apply toany ribosomal RNA and any ribosomal subunit and the sequences of rRNAare well known.

Control of ribosomal activity is thus envisaged through use of thepresent effector protein in concert with a suitable guide to theribosomal target. This may be through cleavage of, or binding to, theribosome. In particular, reduction of ribosomal activity is envisaged.This may be useful in assaying ribosomal function in vivo or in vitro,but also as a means of controlling therapies based on ribosomalactivity, in vivo or in vitro. Furthermore, control (i.e. reduction) ofprotein synthesis in an in vivo or in vitro system is envisaged, suchcontrol including antibiotic and research and diagnostic use.

Riboswitches

A riboswitch (also known as an aptozyme) is a regulatory segment of amessenger RNA molecule that binds a small molecule. This typicallyresults in a change in production of the proteins encoded by the mRNA.Thus, control of riboswitch activity is thus envisaged through use ofthe present effector protein in concert with a suitable guide to theriboswitch target. This may be through cleavage of, or binding to, theriboswitch. In particular, reduction of riboswitch activity isenvisaged. This may be useful in assaying riboswitch function in vivo orin vitro, but also as a means of controlling therapies based onriboswitch activity, in vivo or in vitro. Furthermore, control (i.e.reduction) of protein synthesis in an in vivo or in vitro system isenvisaged. This control, as for rRNA may include antibiotic and researchand diagnostic use.

Ribozymes

Ribozymes are RNA molecules having catalytic properties, analogous toenzymes (which are proteins). As ribozymes, both naturally occurring andengineered, comprise or consist of RNA, they may also be targeted by thepresent RNA-binding effector protein. In some embodiments, the effectorprotein may be a RNA-binding protein cleaves the ribozyme to therebydisable it. Control of ribozymal activity is thus envisaged through useof the present effector protein in concert with a suitable guide to theribozymal target. This may be through cleavage of, or binding to, theribozyme. In particular, reduction of ribozymal activity is envisaged.This may be useful in assaying ribozymal function in vivo or in vitro,but also as a means of controlling therapies based on ribozymalactivity, in vivo or in vitro.

Gene Expression, Including RNA Processing

The effector protein may also be used, together with a suitable guide,to target gene expression, including via control of RNA processing. Thecontrol of RNA processing may include RNA processing reactions such asRNA splicing, including alternative splicing, via targeting of RNApol;viral replication (in particular of satellite viruses, bacteriophagesand retroviruses, such as HBV, HBC and HIV and others listed herein)including virioids in plants; and tRNA biosynthesis. The effectorprotein and suitable guide may also be used to control RNAactivation(RNAa). RNAa leads to the promotion of gene expression, so control ofgene expression may be achieved that way through disruption or reductionof RNAa and thus less promotion of gene expression. This is discussedmore in detail below.

RNAi Screens

Identifying gene products whose knockdown is associated with phenotypicchanges, biological pathways can be interrogated and the constituentparts identified, via RNAi screens. Control may also be exerted over orduring these screens by use of the effector protein and suitable guideto remove or reduce the activity of the RNAi in the screen and thusreinstate the activity of the (previously interfered with) gene product(by removing or reducing the interference/repression).

Satellite RNAs (satRNAs) and satellite viruses may also be treated.

Control herein with reference to RNase activity generally meansreduction, negative disruption or known-down or knock out.

In Vivo RNA Applications Inhibition of Gene Expression

The target-specific RNAses provided herein allow for very specificcutting of a target RNA. The interference at RNA level allows formodulation both spatially and temporally and in a non-invasive way, asthe genome is not modified.

A number of diseases have been demonstrated to be treatable by mRNAtargeting. While most of these studies relate to administration ofsiRNA, it is clear that the RNA targeting effector proteins providedherein can be applied in a similar way.

Examples of mRNA targets (and corresponding disease treatments) areVEGF, VEGF-R1 and RTP801 (in the treatment of AMD and/or DME), Caspase 2(in the treatment of Naion)ADRB2 (in the treatment of intraocularpressure), TRPVI (in the treatment of Dry eye syndrome, Syk kinase (inthe treatment of asthma), Apo B (in the treatment ofhypercholesterolemia), PLK1, KSP and VEGF (in the treatment of solidtumors), Ber-Abl (in the treatment of CMLXBurnett and Rossi Chem Biol.2012, 19(1): 60-71)). Similarly, RNA targeting has been demonstrated tobe effective in the treatment of RNA-virus mediated diseases such as HIV(targeting of HIV Tet and Rev), RSV (targeting of RSV nucleocapsid) andHCV (targeting of miR-122) (Burnett and Rossi Chem Biol. 2012, 19(1):60-71).

It is further envisaged that the RNA targeting effector protein of theinvention can be used for mutation specific or allele specificknockdown. Guide RNA's can be designed that specifically target asequence in the transcribed mRNA comprising a mutation or anallele-specific sequence. Such specific knockdown is particularlysuitable for therapeutic applications relating to disorders associatedwith mutated or allele-specific gene products. For example, most casesof familial hypobetalipoproteinemia (FHBL) are caused by mutations inthe ApoB gene. This gene encodes two versions of the apolipoprotein Bprotein: a short version (ApoB-48) and a longer version (ApoB-100).Several ApoB gene mutations that lead to FHBL cause both versions ofApoB to be abnormally short. Specifically targeting and knockdown ofmutated ApoB mRNA transcripts with an RNA targeting effector protein ofthe invention may be beneficial in treatment of FHBL. As anotherexample, Huntington's disease (HD) is caused by an expansion of CAGtriplet repeats in the gene coding for the Huntingtin protein, whichresults in an abnormal protein. Specifically targeting and knockdown ofmutated or allele-specific mRNA transcripts encoding the Huntingtinprotein with an RNA targeting effector protein of the invention may bebeneficial in treatment of HD.

It is noted that in this context, and more generally for the variousapplications as described herein, the use of a split version of the RNAtargeting effector protein can be envisaged. Indeed, this may not onlyallow increased specificity but may also be advantageous for delivery.The C2c2 is split in the sense that the two parts of the C2c2 enzymesubstantially comprise a functioning C2c2. Ideally, the split shouldalways be so that the catalytic domain(s) are unaffected. That C2c2 mayfunction as a nuclease or it may be a dead-C2c2 which is essentially anRNA-binding protein with very little or no catalytic activity, due totypically mutation(s) in its catalytic domains.

Each half of the split C2c2 may be fused to a dimerization partner. Bymeans of example, and without limitation, employing rapamycin sensitivedimerization domains, allows to generate a chemically inducible splitC2c2 for temporal control of C2c2activity. C2c2 can thus be renderedchemically inducible by being split into two fragments and thatrapamycin-sensitive dimerization domains may be used for controlledreassembly of the C2c2. The two parts of the split C2c2 can be thoughtof as the N′ terminal part and the C′ terminal part of the split C2c2.The fusion is typically at the split point of the C2c2. In other words,the C′ terminal of the N′ terminal part of the split C2c2 is fused toone of the dimer halves, whilst the N′ terminal of the C′ terminal partis fused to the other dimer half.

The C2c2 does not have to be split in the sense that the break is newlycreated. The split point is typically designed in silico and cloned intothe constructs. Together, the two parts of the split C2c2, the N′terminal and C′ terminal parts, form a full C2c2, comprising preferablyat least 70% or more of the wildtype amino acids (or nucleotidesencoding them), preferably at least 80% or more, preferably at least 90%or more, preferably at least 95% or more, and most preferably at least99% or more of the wildtype amino acids (or nucleotides encoding them).Some trimming may be possible, and mutants are envisaged. Non-functionaldomains may be removed entirely. What is important is that the two partsmay be brought together and that the desired C2c2 function is restoredor reconstituted. The dimer may be a homodimer or a heterodimer.

In certain embodiments, the C2c2 effector as described herein may beused for mutation-specific, or allele-specific targeting, such as. formutation-specific, or allele-specific knockdown.

The RNA targeting effector protein can moreover be fused to anotherfunctional RNAse domain, such as a non-specific RNase or Argonaute 2,which acts in synergy to increase the RNAse activity or to ensurefurther degradation of the message.

Modulation of Gene Expression Through Modulation of RNA Function

Apart from a direct effect on gene expression through cleavage of themRNA, RNA targeting can also be used to impact specific aspects of theRNA processing within the cell, which may allow a more subtle modulationof gene expression. Generally, modulation can for instance be mediatedby interfering with binding of proteins to the RNA, such as for instanceblocking binding of proteins, or recruiting RNA binding proteins.Indeed, modulations can be ensured at different levels such as splicing,transport, localization, translation and turnover of the mRNA. Similarlyin the context of therapy, it can be envisaged to address (pathogenic)malfunctioning at each of these levels by using RNA-specific targetingmolecules. In these embodiments it is in many cases preferred that theRNA targeting protein is a “dead” C2c2 that has lost the ability to cutthe RNA target but maintains its ability to bind thereto, such as themutated forms of c2c2 described herein.

a) Alternative Splicing

Many of the human genes express multiple mRNAs as a result ofalternative splicing. Different diseases have been shown to be linked toaberrant splicing leading to loss of function or gain of function of theexpressed gene. While some of these diseases are caused by mutationsthat cause splicing defects, a number of these are not. One therapeuticoption is to target the splicing mechanism directly. The RNA targetingeffector proteins described herein can for instance be used to block orpromote slicing, include or exclude exons and influence the expressionof specific isoforms and/or stimulate the expression of alternativeprotein products. Such applications are described in more detail below.

A RNA targeting effector protein binding to a target RNA can stericallyblock access of splicing factors to the RNA sequence. The RNA targetingeffector protein targeted to a splice site may block splicing at thesite, optionally redirecting splicing to an adjacent site. For instancea RNA targeting effector protein binding to the 5′ splice site bindingcan block the recruitment of the U1 component of the spliceosome,favoring the skipping of that exon. Alternatively, a RNA targetingeffector protein targeted to a splicing enhancer or silencer can preventbinding of transacting regulatory splicing factors at the target siteand effectively block or promote splicing. Exon exclusion can further beachieved by recruitment of ILF2/3 to precursor mRNA near an exon by anRNA targeting effector protein as described herein. As yet anotherexample, a glycine rich domain can be attached for recruitment of hnRNPA1 and exon exclusion (Del Gatto-Konczak et al. Mol Cell Biol. 1999January; 19(1):251-60).

In certain embodiments, through appropriate selection of gRNA, specificsplice variants may be targeted, while other splice variants will not betargeted

In some cases the RNA targeting effector protein can be used to promoteslicing (e.g. where splicing is defective). For instance a RNA targetingeffector protein can be associated with an effector capable ofstabilizing a splicing regulatory stem-loop in order to furthersplicing. The RNA targeting effector protein can be linked to aconsensus binding site sequence for a specific splicing factor in orderto recruit the protein to the target DNA.

Examples of diseases which have been associated with aberrant splicinginclude, but are not limited to Paraneoplastic Opsoclonus MyoclonusAtaxia (or POMA), resulting from a loss of Nova proteins which regulatesplicing of proteins that function in the synapse, and Cystic Fibrosis,which is caused by defective splicing of a cystic fibrosis transmembraneconductance regulator, resulting in the production of nonfunctionalchloride channels. In other diseases aberrant RNA splicing results ingain-of-function. This is the case for instance in myotonic dystrophywhich is caused by a CUG triplet-repeat expansion (from 50 to >1500repeats) in the 3′UTR of an mRNA, causing splicing defects.

The RNA targeting effector protein can be used to include an exon byrecruiting a splicing factor (such as U1) to a 5′ splicing site topromote excision of introns around a desired exon. Such recruitmentcould be mediated trough a fusion with an arginine/serine rich domain,which functions as splicing activator (Gravely B R and Maniatis T, MolCell. 1998 (5):765-71).

It is envisaged that the RNA targeting effector protein can be used toblock the splicing machinery at a desired locus, resulting in preventingexon recognition and the expression of a different protein product. Anexample of a disorder that may treated is Duchenne muscular dystrophy(DMD), which is caused by mutations in the gene encoding for thedystrophin protein. Almost all DMD mutations lead to frameshifts,resulting in impaired dystrophin translation. The RNA targeting effectorprotein can be paired with splice junctions or exonic splicing enhancers(ESEs) thereby preventing exon recognition, resulting in the translationof a partially functional protein. This converts the lethal Duchennephenotype into the less severe Becker phenotype.

b) RNA Modification

RNA editing is a natural process whereby the diversity of gene productsof a given sequence is increased by minor modification in the RNA.Typically, the modification involves the conversion of adenosine (A) toinosine (I), resulting in an RNA sequence which is different from thatencoded by the genome. RNA modification is generally ensured by the ADARenzyme, whereby the pre-RNA target forms an imperfect duplex RNA bybase-pairing between the exon that contains the adenosine to be editedand an intronic non-coding element. A classic example of A-I editing isthe glutamate receptor GluR-B mRNA, whereby the change results inmodified conductance properties of the channel (Higuchi M, et al. Cell.1993; 75:1361-70).

In humans, a heterozygous functional-null mutation in the ADAR1 geneleads to a skin disease, human pigmentary genodermatosis (Miyamura Y, etal. Am J Hum Genet. 2003; 73:693-9). It is envisaged that the RNAtargeting effector proteins of the present invention can be used tocorrect malfunctioning RNA modification.

It is further envisaged that RNA adenosine methylase(N(6)-methyladenosine) can be fused to the RNA targeting effectorproteins of the invention and targeted to a transcript of interest. Thismethylase causes reversible methylation, has regulatory roles and mayaffect gene expression and cell fate decisions by modulating multipleRNA-related cellular pathways (Fu et al Nat Rev Genet. 2014;15(5):293-306).

c) Polyadenylation

Polyadenylation of an mRNA is important for nuclear transport,translation efficiency and stability of the mRNA, and all of these, aswell as the process of polyadenylation, depend on specific RBPs. Mosteukaryotic mRNAs receive a 3′ poly(A) tail of about 200 nucleotidesafter transcription. Polyadenylation involves different RNA-bindingprotein complexes which stimulate the activity of a poly(A)polymerase(Minvielle-Sebastia L et al. Curr Opin Cell Biol. 1999; 11:352-7). It isenvisaged that the RNA-targeting effector proteins provided herein canbe used to interfere with or promote the interaction between theRNA-binding proteins and RNA.

Examples of diseases which have been linked to defective proteinsinvolved in polyadenylation are oculopharyngeal muscular dystrophy(OPMD) (Brais B, et al. Nat Genet. 1998; 18:164-7).

d) RNA Export

After pre-mRNA processing, the mRNA is exported from the nucleus to thecytoplasm. This is ensured by a cellular mechanism which involves thegeneration of a carrier complex, which is then translocated through thenuclear pore and releases the mRNA in the cytoplasm, with subsequentrecycling of the carrier.

Overexpression of proteins (such as TAP) which play a role in the exportof RNA has been found to increase export of transcripts that areotherwise ineffeciently exported in Xenopus (Katahira J, et al. EMBO J.1999; 18:2593-609).

e) mRNA Localization

mRNA localization ensures spatially regulated protein production.Localization of transcripts to a specific region of the cell can beensured by localization elements. In particular embodiments, it isenvisaged that the effector proteins described herein can be used totarget localization elements to the RNA of interest. The effectorproteins can be designed to bind the target transcript and shuttle themto a location in the cell determined by its peptide signal tag. Moreparticularly for instance, a RNA targeting effector protein fused to anuclear localization signal (NLS) can be used to alter RNA localization.

Further examples of localization signals include the zipcode bindingprotein (ZBP1) which ensures localization of β-actin to the cytoplasm inseveral asymmetric cell types, KDEL retention sequence (localization toendoplasmic reticulum), nuclear export signal (localization tocytoplasm), mitochondrial targeting signal (localization tomitochondria), peroxisomal targeting signal (localization to peroxisome)and m6A marking/YTHDF2 (localization to p-bodies). Other approaches thatare envisaged are fusion of the RNA targeting effector protein withproteins of known localization (for instance membrane, synapse).

Alternatively, the effector protein according to the invention may forinstance be used in localization-dependent knockdown. By fusing theeffector protein to a appropriate localization signal, the effector istargeted to a particular cellular compartment. Only target RNAs residingin this compartment will effectively be targeted, whereas otherwiseidentical targets, but residing in a different cellular compartment willnot be targeted, such that a localization dependent knockdown can beestablished.

f) Translation

The RNA targeting effector proteins described herein can be used toenhance or repress translation. It is envisaged that upregulatingtranslation is a very robust way to control cellular circuits. Further,for functional studies a protein translation screen can be favorableover transcriptional upregulation screens, which have the shortcomingthat upregulation of transcript does not translate into increasedprotein production.

It is envisaged that the RNA targeting effector proteins describedherein can be used to bring translation initiation factors, such asEIF4G in the vicinity of the 5′ untranslated repeat (5′UTR) of amessenger RNA of interest to drive translation (as described in DeGregorio et al. EMBO J. 1999; 18(17):4865-74 for a non-reprogrammableRNA binding protein). As another example GLD2, a cytoplasmic poly(A)polymerase, can be recruited to the target mRNA by an RNA targetingeffector protein. This would allow for directed polyadenylation of thetarget mRNA thereby stimulating translation.

Similarly, the RNA targeting effector proteins envisaged herein can beused to block translational repressors of mRNA, such as ZBP1(Huttelmaier S, et al. Nature. 2005; 438:512-5). By binding totranslation initiation site of a target RNA, translation can be directlyaffected.

In addition, fusing the RNA targeting effector proteins to a proteinthat stabilizes mRNAs, e.g. by preventing degradation thereof such asRNase inhibitors, it is possible to increase protein production from thetranscripts of interest.

It is envisaged that the RNA targeting effector proteins describedherein can be used to repress translation by binding in the 5′ UTRregions of a RNA transcript and preventing the ribosome from forming andbeginning translation.

Further, the RNA targeting effector protein can be used to recruit Caf1,a component of the CCR4-NOT deadenylase complex, to the target mRNA,resulting in deadenylation or the target transcript and inhibition ofprotein translation.

For instance, the RNA targeting effector protein of the invention can beused to increase or decrease translation of therapeutically relevantproteins. Examples of therapeutic applications wherein the RNA targetingeffector protein can be used to downregulate or upregulate translationare in amyotrophic lateral sclerosis (ALS) and cardiovascular disorders.Reduced levels of the glial glutamate transporter EAAT2 have beenreported in ALS motor cortex and spinal cord, as well as multipleabnormal EAAT2 mRNA transcripts in ALS brain tissue. Loss of the EAAT2protein and function thought to be the main cause of excitotoxicity inALS. Restoration of EAAT2 protein levels and function may providetherapeutic benefit. Hence, the RNA targeting effector protein can bebeneficially used to upregulate the expression of EAAT2 protein, e.g. byblocking translational repressors or stabilizing mRNA as describedabove. Apolipoprotein A1 is the major protein component of high densitylipoprotein (HDL) and ApoA1 and HDL are generally considered asatheroprotective. It is envisages that the RNA targeting effectorprotein can be beneficially used to upregulate the expression of ApoA1,e.g. by blocking translational repressors or stabilizing mRNA asdescribed above.

g) mRNA Turnover

Translation is tightly coupled to mRNA turnover and regulated mRNAstability. Specific proteins have been described to be involved in thestability of transcripts (such as the ELAV/Hu proteins in neurons, KeeneJ D, 1999, Proc Natl Acad Sci USA. 96:5-7) and tristetraprolin (TTP).These proteins stabilize target mRNAs by protecting the messages fromdegradation in the cytoplasm (Peng S S et al., 1988, EMBO J.17:3461-70).

It can be envisaged that the RNA-targeting effector proteins of thepresent invention can be used to interfere with or to promote theactivity of proteins acting to stabilize mRNA transcripts, such thatmRNA turnover is affected. For instance, recruitment of human TTP to thetarget RNA using the RNA targeting effector protein would allow foradenylate-uridylate-rich element (AU-rich element) mediatedtranslational repression and target degradation. AU-rich elements arefound in the 3′ UTR of many mRNAs that code for proto-oncogenes, nucleartranscription factors, and cytokines and promote RNA stability. Asanother example, the RNA targeting effector protein can be fused to HuR,another mRNA stabilization protein (Hinman M N and Lou H, Cell Mol LifeSci 2008; 65:3168-81), and recruit it to a target transcript to prolongits lifetime or stabilize short-lived mRNA.

It is further envisaged that the RNA-targeting effector proteinsdescribed herein can be used to promote degradation of targettranscripts. For instance, m6A methyltransferase can be recruited to thetarget transcript to localize the transcript to P-bodies leading todegradation of the target.

As yet another example, an RNA targeting effector protein as describedherein can be fused to the non-specific endonuclease domain PilTN-terminus (PIN), to recruit it to a target transcript and allowdegradation thereof.

Patients with paraneoplastic neurological disorder (PND)-associatedencephalomyelitis and neuropathy are patients who develop autoantibodiesagainst Hu-proteins in tumors outside of the central nervous system(Szabo A et al. 1991, Cell; 67:325-33 which then cross the blood-brainbarrier. It can be envisaged that the RNA-targeting effector proteins ofthe present invention can be used to interfere with the binding ofauto-antibodies to mRNA transcripts.

Patients with dystrophy type 1 (DM1), caused by the expansion of (CUG)nin the 3′ UTR of dystrophia myotonica-protein kinase (DMPK) gene, arecharacterized by the accumulation of such transcripts in the nucleus. Itis envisaged that the RNA targeting effector proteins of the inventionfused with an endonuclease targeted to the (CUG)n repeats could inhibitsuch accumulation of aberrant transcripts.

h) Interaction with Multi-Functional Proteins

Some RNA-binding proteins bind to multiple sites on numerous RNAs tofunction in diverse processes. For instance, the hnRNP A1 protein hasbeen found to bind exonic splicing silencer sequences, antagonizing thesplicing factors, associate with telomere ends (thereby stimulatingtelomere activity) and bind miRNA to facilitate Drosha-mediatedprocessing thereby affecting maturation. It is envisaged that theRNA-binding effector proteins of the present invention can interferewith the binding of RNA-binding proteins at one or more locations.

i) RNA Folding

RNA adopts a defined structure in order to perform its biologicalactivities. Transitions in conformation among alternative tertiarystructures are critical to most RNA-mediated processes. However, RNAfolding can be associated with several problems. For instance, RNA mayhave a tendency to fold into, and be upheld in, improper alternativeconformations and/or the correct tertiary structure may not besufficiently thermodynamically favored over alternative structures. TheRNA targeting effector protein, in particular a cleavage-deficient ordead RNA targeting protein, of the invention may be used to directfolding of (m)RNA and/or capture the correct tertiary structure thereof.

Use of RNA-Targeting Effector Protein in Modulating Cellular Status

In certain embodiments C2c2 in a complex with crRNA is activated uponbinding to target RNA and subsequently cleaves any nearby ssRNA targets(i.e. “collateral” or “bystander” effects). C2c2, once primed by thecognate target, can cleave other (non-complementary) RNA molecules. Suchpromiscuous RNA cleavage could potentially cause cellular toxicity, orotherwise affect cellular physiology or cell status.

Accordingly, in certain embodiments, the non-naturally occurring orengineered composition, vector system, or delivery systems as derscribedherein are used for or are for use in induction of cell dormancy. Incertain embodiments, the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as derscribed herein areused for or are for use in induction of cell cycle arrest. In certainembodiments, the non-naturally occurring or engineered composition,vector system, or delivery systems as derscribed herein are used for orare for use in reduction of cell growth and/or cell proliferation, Incertain embodiments, the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as derscribed herein areused for or are for use in induction of cell anergy. In certainembodiments, the non-naturally occurring or engineered composition,vector system, or delivery systems as derscribed herein are used for orare for use in induction of cell apoptosis. In certain embodiments, thenon-naturally occurring or engineered composition, vector system, ordelivery systems as derscribed herein are used for or are for use inincuction of cell necrosis. In certain embodiments, the non-naturallyoccurring or engineered composition, vector system, or delivery systemsas derscribed herein are used for or are for use in induction of celldeath. In certain embodiments, the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as derscribed herein areused for or are for use in induction of programmed cell death.

In certain embodiments, the invention relates to a method for inductionof cell dormancy comprising introducing or inducing the non-naturallyoccurring or engineered composition, vector system, or delivery systemsas derscribed herein. In certain embodiments, the invention relates to amethod for induction of cell cycle arrest comprising introducing orinducing the non-naturally occurring or engineered composition, vectorsystem, or delivery systems as derscribed herein. In certainembodiments, the invention relates to a method for reduction of cellgrowth and/or cell proliferation comprising introducing or inducing thenon-naturally occurring or engineered composition, vector system, ordelivery systems as derscribed herein. In certain embodiments, theinvention relates to a method for induction of cell anergy comprisingintroducing or inducing the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as derscribed herein. Incertain embodiments, the invention relates to a method for induction ofcell apoptosis comprising introducing or inducing the non-naturallyoccurring or engineered composition, vector system, or delivery systemsas derscribed herein. In certain embodiments, the invention relates to amethod for induction of cell necrosis comprising introducing or inducingthe non-naturally occurring or engineered composition, vector system, ordelivery systems as derscribed herein. In certain embodiments, theinvention relates to a method for induction of cell death comprisingintroducing or inducing the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as derscribed herein. Incertain embodiments, the invention relates to a method for induction ofprogrammed cell death comprising introducing or inducing thenon-naturally occurring or engineered composition, vector system, ordelivery systems as derscribed herein.

The methods and uses as described herein may be therapeutic orprophylactic and may target particular cells, cell (sub)populations, orcell/tissue types. In particular, the methods and uses as describedherein may be therapeutic or prophylactic and may target particularcells, cell (sub)populations, or cell/tissue types expressing one ormore target sequences, such as one or more particular target RNA (e.g.ss RNA). Without limitation, target cells may for instance be cancercells expressing a particular transcript, e.g. neurons of a given class,(immune) cells causing e.g. autoimmunity, or cells infected by aspecific (e.g. viral) pathogen, etc.

Accordingly, in certain embodiments, the invention relates to a methodfor treating a pathological condition characterized by the presence ofundersirable cells (host cells), comprising introducing or inducing thenon-naturally occurring or engineered composition, vector system, ordelivery systems as derscribed herein. In certain embodiments, theinvention relates the use of the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as derscribed herein fortreating a pathological condition characterized by the presence ofundersirable cells (host cells). In certain embodiments, the inventionrelates the non-naturally occurring or engineered composition, vectorsystem, or delivery systems as derscribed herein for use in treating apathological condition characterized by the presence of undersirablecells (host cells). It is to be understood that preferably theCRISPR-Cas system targets a target specific for the undesirable cells.In certain embodiments, the invention relates to the use of thenon-naturally occurring or engineered composition, vector system, ordelivery systems as derscribed herein for treating, preventing, oralleviating cancer. In certain embodiments, the invention relates to thenon-naturally occurring or engineered composition, vector system, ordelivery systems as derscribed herein for use in treating, preventing,or alleviating cancer. In certain embodiments, the invention relates toa method for treating, preventing, or alleviating cancer comprisingintroducing or inducing the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as derscribed herein. Itis to be understood that preferably the CRISPR-Cas system targets atarget specific for the cancer cells. In certain embodiments, theinvention relates to the use of the non-naturally occurring orengineered composition, vector system, or delivery systems as derscribedherein for treating, preventing, or alleviating infection of cells by apathogen. In certain embodiments, the invention relates to thenon-naturally occurring or engineered composition, vector system, ordelivery systems as derscribed herein for use in treating, preventing,or alleviating infection of cells by a pathogen. In certain embodiments,the invention relates to a method for treating, preventing, oralleviating infection of cells by a pathogen comprising introducing orinducing the non-naturally occurring or engineered composition, vectorsystem, or delivery systems as derscribed herein. It is to be understoodthat preferably the CRISPR-Cas system targets a target specific for thecells infected by the pathogen (e.g. a pathogen derived target). Incertain embodiments, the invention relates to the use of thenon-naturally occurring or engineered composition, vector system, ordelivery systems as derscribed herein for treating, preventing, oralleviating an autoimmune disorder. In certain embodiments, theinvention relates to the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as derscribed herein foruse in treating, preventing, or alleviating an autoimmune disorder. Incertain embodiments, the invention relates to a method for treating,preventing, or alleviating an autoimmune disorder comprising introducingor inducing the non-naturally occurring or engineered composition,vector system, or delivery systems as derscribed herein. It is to beunderstood that preferably the CRISPR-Cas system targets a targetspecific for the cells responsible for the autoimmune disorder (e.g.specific immune cells).

Use of RNA-Targeting Effector Protein in RNA Detection

It is further envisaged that the RNA targeting effector protein can beused in Northern blot assays. Northern blotting involves the use ofelectrophoresis to separate RNA samples by size. The RNA targetingeffector protein can be used to specifically bind and detect the targetRNA sequence.

A RNA targeting effector protein can be fused to a fluorescent protein(such as GFP) and used to track RNA localization in living cells. Moreparticularly, the RNA targeting effector protein can be inactivated inthat it no longer cleaves RNA. In particular embodiments, it isenvisaged that a split RNA targeting effector protein can be used,whereby the signal is dependent on the binding of both subproteins, inorder to ensure a more precise visualization. Alternatively, a splitfluorescent protein can be used that is reconstituted when multiple RNAtargeting effector protein complexes bind to the target transcript. Itis further envisaged that a transcript is targeted at multiple bindingsites along the mRNA so the fluorescent signal can amplify the truesignal and allow for focal identification. As yet another alternative,the fluorescent protein can be reconstituted form a split intein.

RNA targeting effector proteins are for instance suitably used todetermine the localization of the RNA or specific splice variants, thelevel of mRNA transcript, up- or down-regulation of transcripts anddisease-specific diagnosis. The RNA targeting effector proteins can beused for visualization of RNA in (living) cells using e.g. fluorescentmicroscopy or flow cytometry, such as fluorescence-activated cellsorting (FACS) which allows for high-throughput screening of cells andrecovery of living cells following cell sorting. Further, expressionlevels of different transcripts can be assessed simultaneously understress, e.g. inhibition of cancer growth using molecular inhibitors orhypoxic conditions on cells. Another application would be to tracklocalization of transcripts to synaptic connections during a neuralstimulus using two photon microscopy.

In certain embodiments, the components or complexes according to theinvention as described herein can be used in multiplexed error-robustfluorescence in situ hybridization (MERFISH; Chen et al. Science; 2015;348(6233)), such as for instance with (fluorescently) labeled C2c2effectors.

In Vitro Apex Labeling

Cellular processes depend on a network of molecular interactions amongprotein, RNA, and DNA. Accurate detection of protein-DNA and protein-RNAinteractions is key to understanding such processes. In vitro proximitylabeling technology employs an affinity tag combined with e.g. aphotoactivatable probe to label polypeptides and RNAs in the vicinity ofa protein or RNA of interest in vitro. After UV irradiation thephotoactivatable group reacts with proteins and other molecules that arein close proximity to the tagged molecule, thereby labelling them.Labelled interacting molecules can subsequently be recovered andidentified. The RNA targeting effector protein of the invention can forinstance be used to target a probe to a selected RNA sequence.

These applications could also be applied in animal models for in vivoimaging of disease relevant applications or difficult-to culture celltypes. Use of RNA-targeting effector protein in RNA origami/in vitroassembly lines—combinatorics RNA origami refers to nanoscale foldedstructures for creating two-dimensional or three-dimensional structuresusing RNA as integrated template. The folded structure is encoded in theRNA and the shape of the resulting RNA is thus determined by thesynthesized RNA sequence (Geary, et al. 2014. Science, 345 (6198). pp.799-804). The RNA origami may act as scaffold for arranging othercomponents, such as proteins, into complexes. The RNA targeting effectorprotein of the invention can for instance be used to target proteins ofinterest to the RNA origami using a suitable guide RNA.

These applications could also be applied in animal models for in vivoimaging of disease relevant applications or difficult-to culture celltypes.

Use of RNA-Targeting Effector Protein in RNA Isolation or Purification,Enrichment or Deletion

It is further envisages that the RNA targeting effector protein whencomplexed to RNA can be used to isolate and/or purify the RNA. The RNAtargeting effector protein can for instance be fused to an affinity tagthat can be used to isolate and/or purify the RNA-RNA targeting effectorprotein complex. Such applications are for instance useful in theanalysis of gene expression profiles in cells.

In particular embodiments, it can be envisaged that the RNA targetingeffector proteins can be used to target a specific noncoding RNA (ncRNA)thereby blocking its activity, providing a useful functional probe. Incertain embodiments, the effector protein as described herein may beused to specifically enrich for a particular RNA (including but notlimited to increasing stability, etc.), or alternatively to specificallydeplete a particular RNA (such as without limitation for instanceparticular splice variants, isoforms, etc.).

Interrogation of lincRNA Function and Other Nuclear RNAs

Current RNA knockdown strategies such as siRNA have the disadvantagethat they are mostly limited to targeting cytosolic transcripts sincethe protein machinery is cytosolic. The advantage of a RNA targetingeffector protein of the present invention, an exogenous system that isnot essential to cell function, is that it can be used in anycompartment in the cell. By fusing a NLS signal to the RNA targetingeffector protein, it can be guided to the nucleus, allowing nuclear RNAsto be targeted. It is for instance envisaged to probe the function oflincRNAs. Long intergenic non-coding RNAs (lincRNAs) are a vastlyunderexplored area of research. Most lincRNAs have as of yet unknownfunctions which could be studies using the RNA targeting effectorprotein of the invention.

Identification of RNA Binding Proteins

Identifying proteins bound to specific RNAs can be useful forunderstanding the roles of many RNAs. For instance, many lincRNAsassociate with transcriptional and epigenetic regulators to controltranscription. Understanding what proteins bind to a given lincRNA canhelp elucidate the components in a given regulatory pathway. A RNAtargeting effector protein of the invention can be designed to recruit abiotin ligase to a specific transcript in order to label locally boundproteins with biotin. The proteins can then be pulled down and analyzedby mass spectrometry to identify them.

Assembly of Complexes on RNA and Substrate Shuttling

RNA targeting effector proteins of the invention can further be used toassemble complexes on RNA. This can be achieved by functionalizing theRNA targeting effector protein with multiple related proteins (e.g.components of a particular synthesis pathway). Alternatively, multipleRNA targeting effector proteins can be functionalized with suchdifferent related proteins and targeted to the same or adjacent targetRNA. Useful application of assembling complexes on RNA are for instancefacilitating substrate shuttling between proteins.

Synthetic Biology

The development of biological systems have a wide utility, including inclinical applications. It is envisaged that the programmable RNAtargeting effector proteins of the invention can be used fused to splitproteins of toxic domains for targeted cell death, for instance usingcancer-linked RNA as target transcript. Further, pathways involvingprotein-protein interaction can be influenced in synthetic biologicalsystems with e.g. fusion complexes with the appropriate effectors suchas kinases or other enzymes.

Protein Splicing: Inteins

Protein splicing is a post-translational process in which an interveningpolypeptide, referred to as an intein, catalyzes its own excision fromthe polypeptides flacking it, referred to as exteins, as well assubsequent ligation of the exteins. The assembly of two or more RNAtargeting effector proteins as described herein on a target transcriptcould be used to direct the release of a split intein (Topilina andMills Mob DNA. 2014 Feb. 4; 5(1):5), thereby allowing for directcomputation of the existence of a mRNA transcript and subsequent releaseof a protein product, such as a metabolic enzyme or a transcriptionfactor (for downstream actuation of transcription pathways). Thisapplication may have significant relevance in synthetic biology (seeabove) or large-scale bioproduction (only produce product under certainconditions).

Inducible, Dosed and Self-Inactivating Systems

In one embodiment, fusion complexes comprising an RNA targeting effectorprotein of the invention and an effector component are designed to beinducible, for instance light inducible or chemically inducible. Suchinducibility allows for activation of the effector component at adesired moment in time.

Light inducibility is for instance achieved by designing a fusioncomplex wherein CRY2 PHR/CIBN pairing is used for fusion. This system isparticularly useful for light induction of protein interactions inliving cells (Konermann S, et al. Nature. 2013; 500:472-476).

Chemical inducibility is for instance provided for by designing a fusioncomplex wherein FKBP/FRB (FK506 binding protein/FKBP rapamycin binding)pairing is used for fusion. Using this system rapamycin is required forbinding of proteins (Zetsche et al. Nat Biotechnol. 2015; 33(2):139-42describes the use of this system for Cas9).

Further, when introduced in the cell as DNA, the RNA targeting effectorprotein of the inventions can be modulated by inducible promoters, suchas tetracycline or doxycycline controlled transcriptional activation(Tet-On and Tet-Off expression system), hormone inducible geneexpression system such as for instance an ecdysone inducible geneexpression system and an arabinose-inducible gene expression system.When delivered as RNA, expression of the RNA targeting effector proteincan be modulated via a riboswitch, which can sense a small molecule liketetracycline (as described in Goldfless et al. Nucleic Acids Res. 2012;40(9):e64).

In one embodiment, the delivery of the RNA targeting effector protein ofthe invention can be modulated to change the amount of protein or crRNAin the cell, thereby changing the magnitude of the desired effect or anyundesired off-target effects.

In one embodiment, the RNA targeting effector proteins described hereincan be designed to be self-inactivating. When delivered to a cell asRNA, either mRNA or as a replication RNA therapeutic (Wrobleska et alNat Biotechnol. 2015 August; 33(8): 839-841), they can self-inactivateexpression and subsequent effects by destroying the own RNA, therebyreducing residency and potential undesirable effects.

For further in vivo applications of RNA targeting effector proteins asdescribed herein, reference is made to Mackay J P et al (Nat Struct MolBiol. 2011 March; 18(3):256-61), Nelles et al (Bioessays. 2015 July;37(7):732-9) and Abil Z and Zhao H (Mol Biosyst. 2015 October;11(10):2658-65), which are incorporated herein by reference. Inparticular, the following applications are envisaged in certainembodiments of the invention, preferably in certain embodiments by usingcatalytically inactive C2c2: enhancing translation (e.g.C2c2—translation promotion factor fusions (e.g. eIF4 fusions));repressing translation (e.g. gRNA targeting ribosome binding sites);exon skipping (e.g. gRNAs targeting splice donor and/or acceptor sites);exon inclusion (e.g. gRNA targeting a particular exon splice donorand/or acceptor site to be included or C2c2 fused to or recruitingspliceosome components (e.g. U1 snRNA)); accessing RNA localization(e.g. C2c2—marker fusions (e.g. EGFP fusions)); altering RNAlocalization (e.g. C2c2—localization signal fusions (e.g. NLS or NESfusions)); RNA degradation (in this case no catalytically inactive C2c2is to be used if relied on the activity of C2c2, alternatively and forincreased specificity, a split C2c2 may be used); inhibition ofnon-coding RNA function (e.g. miRNA), such as by degradation or bindingof gRNA to functional sites (possibly titrating out at specific sites byrelocalization by C2c2-signal sequence fusions).

As described herein before and demonstrated in the Examples, C2c2function is robust to 5′ or 3′ extensions of the crRNA and to extensionof the crRNA loop. It is therefore envisages that MS2 loops and otherrecruitment domains can be added to the crRNA without affecting complexformation and binding to target transcripts. Such modifications to thecrRNA for recruitment of various effector domains are applicable in theuses of a RNA targeted effector proteins described above.

As demonstrated in the Examples, C2c2, in particular LshC2c2, is capableof mediating resistance to RNA phages. It is therefore envisaged thatC2c2 can be used to immunize, e.g. animals, humans and plants, againstRNA-only pathogens, including but not limited to Ebola virus and Zikavirus.

The present inventors have shown that C2c2 can processes (cleaves) itsown array. This applies to both the wildtype C2c2 protein and themutated C2c2 protein containing one or more mutated amino acid residuesR597, H602, R1278 and H1283, such as one or more of the modificationsselected from R597A, H602A, R1278A and H1283A. It is therefore envisagedthat multiple crRNAs designed for different target transcripts and/orapplications can be delivered as a single pre-crRNA or as a singletranscript driven by one promotor. Such method of delivery has theadvantages that it is substantially more compact, easier to synthesizeand easier to delivery in viral systems. Preferably, amino acidnumbering as described herein refers to Lsh C2c2 protein. It will beunderstood that exact amino acid positions may vary for orthologues ofLsh C2c2, which can be adequately determined by protein alignment, as isknown in the art, and as described herein elsewhere.

Aspects of the invention also encompass methods and uses of thecompositions and systems described herein in genome engineering, e.g.for altering or manipulating the expression of one or more genes or theone or more gene products, in prokaryotic or eukaryotic cells, in vitro,in vivo or ex vivo.

In an aspect, the invention provides methods and compositions formodulating, e.g., reducing, expression of a target RNA in cells. In thesubject methods, a C2c2 system of the invention is provided thatinterferes with transcription, stability, and/or translation of an RNA.

In certain embodiments, an effective amount of C2c2 system is used tocleave RNA or otherwise inhibit RNA expression. In this regard, thesystem has uses similar to siRNA and shRNA, thus can also be substitutedfor such methods. The method includes, without limitation, use of a C2c2system as a substitute for e.g., an interfering ribonucleic acid (suchas an siRNA or shRNA) or a transcription template thereof, e.g., a DNAencoding an shRNA. The C2c2 system is introduced into a target cell,e.g., by being administered to a mammal that includes the target cell.

Advantageously, a C2c2 system of the invention is specific. For example,whereas interfering ribonucleic acid (such as an siRNA or shRNA)polynucleotide systems are plagued by design and stability issues andoff-target binding, a C2c2 system of the invention can be designed withhigh specificity.

Destabilized C2c2

In certain embodiments, the effector protein (CRISPR enzyme; C2c2)according to the invention as described herein is associated with orfused to a destabilization domain (DD). In some embodiments, the DD isER50. A corresponding stabilizing ligand for this DD is, in someembodiments, 4HT. As such, in some embodiments, one of the at least oneDDs is ER50 and a stabilizing ligand therefor is 4HT or CMP8. In someembodiments, the DD is DHFR50. A corresponding stabilizing ligand forthis DD is, in some embodiments, TMP. As such, in some embodiments, oneof the at least one DDs is DHFR50 and a stabilizing ligand therefor isTMP. In some embodiments, the DD is ER50. A corresponding stabilizingligand for this DD is, in some embodiments, CMP8. CMP8 may therefore bean alternative stabilizing ligand to 4HT in the ER50 system. While itmay be possible that CMP8 and 4HT can/should be used in a competitivematter, some cell types may be more susceptible to one or the other ofthese two ligands, and from this disclosure and the knowledge in the artthe skilled person can use CMP8 and/or 4HT.

In some embodiments, one or two DDs may be fused to the N-terminal endof the CRISPR enzyme with one or two DDs fused to the C-terminal of theCRISPR enzyme. In some embodiments, the at least two DDs are associatedwith the CRISPR enzyme and the DDs are the same DD, i.e. the DDs arehomologous. Thus, both (or two or more) of the DDs could be ER50 DDs.This is preferred in some embodiments. Alternatively, both (or two ormore) of the DDs could be DHFR50 DDs. This is also preferred in someembodiments. In some embodiments, the at least two DDs are associatedwith the CRISPR enzyme and the DDs are different DDs, i.e. the DDs areheterologous. Thus, one of the DDS could be ER50 while one or more ofthe DDs or any other DDs could be DHFR50. Having two or more DDs whichare heterologous may be advantageous as it would provide a greater levelof degradation control. A tandem fusion of more than one DD at the N orC-term may enhance degradation; and such a tandem fusion can be, forexample ER50-ER50-C2c2 or DHFR-DHFR-C2c2 It is envisaged that highlevels of degradation would occur in the absence of either stabilizingligand, intermediate levels of degradation would occur in the absence ofone stabilizing ligand and the presence of the other (or another)stabilizing ligand, while low levels of degradation would occur in thepresence of both (or two of more) of the stabilizing ligands. Controlmay also be imparted by having an N-terminal ER50 DD and a C-terminalDHFR50 DD.

In some embodiments, the fusion of the CRISPR enzyme with the DDcomprises a linker between the DD and the CRISPR enzyme. In someembodiments, the linker is a GlySer linker. In some embodiments, theDD-CRISPR enzyme further comprises at least one Nuclear Export Signal(NES). In some embodiments, the DD-CRISPR enzyme comprises two or moreNESs. In some embodiments, the DD-CRISPR enzyme comprises at least oneNuclear Localization Signal (NLS). This may be in addition to an NES. Insome embodiments, the CRISPR enzyme comprises or consists essentially ofor consists of a localization (nuclear import or export) signal as, oras part of, the linker between the CRISPR enzyme and the DD. HA or Flagtags are also within the ambit of the invention as linkers. Applicantsuse NLS and/or NES as linker and also use Glycine Serine linkers asshort as GS up to (GGGGS)3.

Destabilizing domains have general utility to confer instability to awide range of proteins; see, e.g., Miyazaki, J Am Chem Soc. Mar. 7,2012; 134(9): 3942-3945, incorporated herein by reference. CMP8 or4-hydroxytamoxifen can be destabilizing domains. More generally, Atemperature-sensitive mutant of mammalian DHFR (DHFRts), a destabilizingresidue by the N-end rule, was found to be stable at a permissivetemperature but unstable at 37° C. The addition of methotrexate, ahigh-affinity ligand for mammalian DHFR, to cells expressing DHFRtsinhibited degradation of the protein partially. This was an importantdemonstration that a small molecule ligand can stabilize a proteinotherwise targeted for degradation in cells. A rapamycin derivative wasused to stabilize an unstable mutant of the FRB domain of mTOR (FRB*)and restore the function of the fused kinase, GSK-3β.6,7 This systemdemonstrated that ligand-dependent stability represented an attractivestrategy to regulate the function of a specific protein in a complexbiological environment. A system to control protein activity can involvethe DD becoming functional when the ubiquitin complementation occurs byrapamycin induced dimerization of FK506-binding protein and FKBP12.Mutants of human FKBP12 or ecDHFR protein can be engineered to bemetabolically unstable in the absence of their high-affinity ligands,Shield-1 or trimethoprim (TMP), respectively. These mutants are some ofthe possible destabilizing domains (DDs) useful in the practice of theinvention and instability of a DD as a fusion with a CRISPR enzymeconfers to the CRISPR protein degradation of the entire fusion proteinby the proteasome. Shield-1 and TMP bind to and stabilize the DD in adose-dependent manner. The estrogen receptor ligand binding domain(ERLBD, residues 305-549 of ERS1) can also be engineered as adestabilizing domain. Since the estrogen receptor signaling pathway isinvolved in a variety of diseases such as breast cancer, the pathway hasbeen widely studied and numerous agonist and antagonists of estrogenreceptor have been developed. Thus, compatible pairs of ERLBD and drugsare known. There are ligands that bind to mutant but not wild-type formsof the ERLBD. By using one of these mutant domains encoding threemutations (L384M, M421G, G521R)12, it is possible to regulate thestability of an ERLBD-derived DD using a ligand that does not perturbendogenous estrogen-sensitive networks. An additional mutation (Y537S)can be introduced to further destabilize the ERLBD and to configure itas a potential DD candidate. This tetra-mutant is an advantageous DDdevelopment. The mutant ERLBD can be fused to a CRISPR enzyme and itsstability can be regulated or perturbed using a ligand, whereby theCRISPR enzyme has a DD. Another DD can be a 12-kDa (107-amino-acid) tagbased on a mutated FKBP protein, stabilized by Shield1 ligand; see,e.g., Nature Methods 5, (2008). For instance a DD can be a modifiedFK506 binding protein 12 (FKBP12) that binds to and is reversiblystabilized by a synthetic, biologically inert small molecule, Shield-1;see, e.g., Banaszynski L A, Chen L C, Maynard-Smith L A, Ooi A G,Wandless T J. A rapid, reversible, and tunable method to regulateprotein function in living cells using synthetic small molecules. Cell.2006; 126:995-1004; Banaszynski L A, Sellmyer M A, Contag C H, WandlessT J, Thorne S H. Chemical control of protein stability and function inliving mice. Nat Med. 2008; 14:1123-1127; Maynard-Smith L A, Chen L C,Banaszynski L A, Ooi A G, Wandless T J. A directed approach forengineering conditional protein stability using biologically silentsmall molecules. The Journal of biological chemistry. 2007;282:24866-24872; and Rodriguez, Chem Biol. Mar. 23, 2012; 19(3):391-398-all of which are incorporated herein by reference and may beemployed in the practice of the invention in selected a DD to associatewith a CRISPR enzyme in the practice of this invention. As can be seen,the knowledge in the art includes a number of DDs, and the DD can beassociated with, e.g., fused to, advantageously with a linker, to aCRISPR enzyme, whereby the DD can be stabilized in the presence of aligand and when there is the absence thereof the DD can becomedestabilized, whereby the CRISPR enzyme is entirely destabilized, or theDD can be stabilized in the absence of a ligand and when the ligand ispresent the DD can become destabilized; the DD allows the CRISPR enzymeand hence the CRISPR-Cas complex or system to be regulated orcontrolled-turned on or off so to speak, to thereby provide means forregulation or control of the system, e.g., in an in vivo or in vitroenvironment. For instance, when a protein of interest is expressed as afusion with the DD tag, it is destabilized and rapidly degraded in thecell, e.g., by proteasomes. Thus, absence of stabilizing ligand leads toa D associated Cas being degraded. When a new DD is fused to a proteinof interest, its instability is conferred to the protein of interest,resulting in the rapid degradation of the entire fusion protein. Peakactivity for Cas is sometimes beneficial to reduce off-target effects.Thus, short bursts of high activity are preferred. The present inventionis able to provide such peaks. In some senses the system is inducible.In some other senses, the system repressed in the absence of stabilizingligand and de-repressed in the presence of stabilizing ligand.

Application of RNA Targeting-CRISPR System to Plants and YeastDefinitions

In general, the term “plant” relates to any various photosynthetic,eukaryotic, unicellular or multicellular organism of the kingdom Plantaecharacteristically growing by cell division, containing chloroplasts,and having cell walls comprised of cellulose. The term plant encompassesmonocotyledonous and dicotyledonous plants. Specifically, the plants areintended to comprise without limitation angiosperm and gymnosperm plantssuch as acacia, alfalfa, amaranth, apple, apricot, artichoke, ash tree,asparagus, avocado, banana, barley, beans, beet, birch, beech,blackberry, blueberry, broccoli, Brussel's sprouts, cabbage, canola,cantaloupe, carrot, cassava, cauliflower, cedar, a cereal, celery,chestnut, cherry, Chinese cabbage, citrus, clementine, clover, coffee,corn, cotton, cowpea, cucumber, cypress, eggplant, elm, endive,eucalyptus, fennel, figs, fir, geranium, grape, grapefruit, groundnuts,ground cherry, gum hemlock, hickory, kale, kiwifruit, kohlrabi, larch,lettuce, leek, lemon, lime, locust, pine, maidenhair, maize, mango,maple, melon, millet, mushroom, mustard, nuts, oak, oats, oil palm,okra, onion, orange, an ornamental plant or flower or tree, papaya,palm, parsley, parsnip, pea, peach, peanut, pear, peat, pepper,persimmon, pigeon pea, pine, pineapple, plantain, plum, pomegranate,potato, pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye,sorghum, safflower, sallow, soybean, spinach, spruce, squash,strawberry, sugar beet, sugarcane, sunflower, sweet potato, sweet corn,tangerine, tea, tobacco, tomato, trees, triticale, turf grasses,turnips, vine, walnut, watercress, watermelon, wheat, yams, yew, andzucchini. The term plant also encompasses Algae, which are mainlyphotoautotrophs unified primarily by their lack of roots, leaves andother organs that characterize higher plants.

The methods for modulating gene expression using the RNA targetingsystem as described herein can be used to confer desired traits onessentially any plant. A wide variety of plants and plant cell systemsmay be engineered for the desired physiological and agronomiccharacteristics described herein using the nucleic acid constructs ofthe present disclosure and the various transformation methods mentionedabove. In preferred embodiments, target plants and plant cells forengineering include, but are not limited to, those monocotyledonous anddicotyledonous plants, such as crops including grain crops (e.g., wheat,maize, rice, millet, barley), fruit crops (e.g., tomato, apple, pear,strawberry, orange), forage crops (e.g., alfalfa), root vegetable crops(e.g., carrot, potato, sugar beets, yam), leafy vegetable crops (e.g.,lettuce, spinach); flowering plants (e.g., petunia, rose,chrysanthemum), conifers and pine trees (e.g., pine fir, spruce); plantsused in phytoremediation (e.g., heavy metal accumulating plants); oilcrops (e.g., sunflower, rape seed) and plants used for experimentalpurposes (e.g., Arabidopsis). Thus, the methods and CRISPR-Cas systemscan be used over a broad range of plants, such as for example withdicotyledonous plants belonging to the orders Magniolales, Illiciales,Laurales, Piperales, Aristochiales, Nymphaeales, Ranunculales,Papeverales, Sarraceniaceae, Trochodendrales, Hamamelidales, Eucomiales,Leitneriales, Myricales, Fagales, Casuarinales, Caryophyllales, Batales,Polygonales, Plumbaginales, Dilleniales, Theales, Malvales, Urticales,Lecythidales, Violales, Salicales, Capparales, Ericales, Diapensales,Ebenales, Primulales, Rosales, Fabales, Podostemales, Haloragales,Myrtales, Cornales, Proteales, San tales, Rafflesiales, Celastrales,Euphorbiales, Rhamnales, Sapindales, Juglandales, Geraniales,Polygalales, Umbellales, Gentianales, Polemoniales, Lamiales,Plantaginales, Scrophulariales, Campanulales, Rubiales, Dipsacales, andAsterales; the methods and CRISPR-Cas systems can be used withmonocotyledonous plants such as those belonging to the ordersAlismatales, Hydrocharitales, Najadales, Triuridales, Commelinales,Eriocaulales, Restionales, Poales, Juncales, Cyperales, Typhales,Bromeliales, Zingiberales, Arecales, Cyclanthales, Pandanales, Arales,Lilliales, and Orchid ales, or with plants belonging to Gymnospermae,e.g those belonging to the orders Pinales, Ginkgoales, Cycadales,Araucariales, Cupressales and Gnetales.

The RNA targeting CRISPR systems and methods of use described herein canbe used over a broad range of plant species, included in thenon-limitative list of dicot, monocot or gymnosperm genera hereunder:Atropa, Alseodaphne, Anacardium, Arachis, Beilschmiedia, Brassica,Carthamus, Cocculus. Croton, Cucumis, Citrus, Citrullus, Capsicum,Catharanthus, Cocos, Coffea, Cucurbila, Daucus, Duguelia, Eschscholzia,Ficus, Fragaria, Glaucium, Glycine, Gossypium, Helianthus. Hevea,Hyoscyamus, Lactuca, Landolphia, Limm, Litsea. Lvycopersicon, Lupimis,Manihot, Majorana, Malus, Medicago. Nicoliana. Olea, Parthenium,Papaver. Persea, Phaseolus, Pistacia, Pisum, Pyrus, Prumis, Raphanus,Ricinus, Senecio, Sinomenium, Stephania, Sinapis, Solanum, Theobroma,Trifolium, Trigonella, Vicia, Vinca, Vilis, and Vigna; and the generaAllium, Andropogon, Aragrostis, Asparagus, Avena, Cynodon, Elaeis,Festuca, Festulolium, Heterocallis, Hordeum, Lemna, Lolium, Musa, Oryra,Panicum, Pannesetum, Phleum, Poa, Secale. Sorghum, Triticum, Zea, Abies,Cunninghamia, Ephedra, Picea, Pinus, and Pseudotsuga.

The RNA targeting CRISPR systems and methods of use can also be usedover a broad range of “algae” or “algae cells”; including for examplealgea selected from several eukaryotic phyla, including the Rhodophyta(red algae), Chlorophyta (green algae), Phaeophyta (brown algae),Bacillariophyta (diatoms), Eustigmatophyta and dinoflagellates as wellas the prokaryotic phylum Cyanobacteria (blue-green algae). The term“algae” includes for example algae selected from: Amphora, Anabaena,Anikstrodesmis, Botryococcus, Chaetoceros, Chlamydomonas, Chlorella,Chlorococcum, Cyclotella, Cylindrotheca, Dunaliella, Emiliana, Euglena,Hematococcus, Isochrysis, Monochrysis, Monoraphidium, Nannochloris,Nannnochloropsis, Navicula, Nephrochloris, Nephroselmis, Nitzschia,Nodularia, Nostoc, Oochromonas, Oocystis, Oscillartoria, Pavlova,Phaeodactylum, Playtmonas, Pleurochrysis, Porhyra, Pseudoanabaena,Pyramimonas, Stichococcus, Synechococcus, Synechocystis, Tetraselmis,Thalassiosira, and Trichodesmium.

A part of a plant, i.e., a “plant tissue” may be treated according tothe methods of the present invention to produce an improved plant. Planttissue also encompasses plant cells. The term “plant cell” as usedherein refers to individual units of a living plant, either in an intactwhole plant or in an isolated form grown in in vitro tissue cultures, onmedia or agar, in suspension in a growth media or buffer or as a part ofhigher organized unites, such as, for example, plant tissue, a plantorgan, or a whole plant.

A “protoplast” refers to a plant cell that has had its protective cellwall completely or partially removed using, for example, mechanical orenzymatic means resulting in an intact biochemical competent unit ofliving plant that can reform their cell wall, proliferate and regenerategrow into a whole plant under proper growing conditions.

The term “transformation” broadly refers to the process by which a planthost is genetically modified by the introduction of DNA by means ofAgrobacteria or one of a variety of chemical or physical methods. Asused herein, the term “plant host” refers to plants, including anycells, tissues, organs, or progeny of the plants. Many suitable planttissues or plant cells can be transformed and include, but are notlimited to, protoplasts, somatic embryos, pollen, leaves, seedlings,stems, calli, stolons, microtubers, and shoots. A plant tissue alsorefers to any clone of such a plant, seed, progeny, propagule whethergenerated sexually or asexually, and descendents of any of these, suchas cuttings or seed.

The term “transformed” as used herein, refers to a cell, tissue, organ,or organism into which a foreign DNA molecule, such as a construct, hasbeen introduced. The introduced DNA molecule may be integrated into thegenomic DNA of the recipient cell, tissue, organ, or organism such thatthe introduced DNA molecule is transmitted to the subsequent progeny. Inthese embodiments, the “transformed” or “transgenic” cell or plant mayalso include progeny of the cell or plant and progeny produced from abreeding program employing such a transformed plant as a parent in across and exhibiting an altered phenotype resulting from the presence ofthe introduced DNA molecule. Preferably, the transgenic plant is fertileand capable of transmitting the introduced DNA to progeny through sexualreproduction.

The term “progeny”, such as the progeny of a transgenic plant, is onethat is born of, begotten by, or derived from a plant or the transgenicplant. The introduced DNA molecule may also be transiently introducedinto the recipient cell such that the introduced DNA molecule is notinherited by subsequent progeny and thus not considered “transgenic”.Accordingly, as used herein, a “non-transgenic” plant or plant cell is aplant which does not contain a foreign DNA stably integrated into itsgenome.

The term “plant promoter” as used herein is a promoter capable ofinitiating transcription in plant cells, whether or not its origin is aplant cell. Exemplary suitable plant promoters include, but are notlimited to, those that are obtained from plants, plant viruses, andbacteria such as Agrobacterium or Rhizobium which comprise genesexpressed in plant cells.

As used herein, a “fungal cell” refers to any type of eukaryotic cellwithin the kingdom of fungi. Phyla within the kingdom of fungi includeAscomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota,Glomeromycota, Microsporidia, and Neocallimastigomycota. Fungal cellsmay include yeasts, molds, and filamentous fungi. In some embodiments,the fungal cell is a yeast cell.

As used herein, the term “yeast cell” refers to any fungal cell withinthe phyla Ascomycota and Basidiomycota. Yeast cells may include buddingyeast cells, fission yeast cells, and mold cells. Without being limitedto these organisms, many types of yeast used in laboratory andindustrial settings are part of the phylum Ascomycota. In someembodiments, the yeast cell is an S. cerervisiae, Kluyveromycesmarxianus, or Issatchenkia orientalis cell. Other yeast cells mayinclude without limitation Candida spp. (e.g., Candida albicans),Yarrowia spp. (e.g., Yarrowia lipolytica), Pichia spp. (e.g., Pichiapastoris), Kluyveromyces spp. (e.g., Kluyveromyces lactis andKluyveromyces marxianus), Neurospora spp. (e.g., Neurospora crassa),Fusarium spp. (e.g., Fusarium oxysporum), and Issatchenkia spp. (e.g.,Issatchenkia orientalis, a.k.a. Pichia kudriavzevii and Candidaacidothermophilum). In some embodiments, the fungal cell is afilamentous fungal cell. As used herein, the term “filamentous fungalcell” refers to any type of fungal cell that grows in filaments, i.e.,hyphae or mycelia. Examples of filamentous fungal cells may includewithout limitation Aspergillus spp. (e.g., Aspergillus niger),Trichoderma spp. (e.g., Trichoderma reesei), Rhizopus spp. (e.g.,Rhizopus oryzae), and Mortierella spp. (e.g., Mortierella isabellina).

In some embodiments, the fungal cell is an industrial strain. As usedherein, “industrial strain” refers to any strain of fungal cell used inor isolated from an industrial process, e.g., production of a product ona commercial or industrial scale. Industrial strain may refer to afungal species that is typically used in an industrial process, or itmay refer to an isolate of a fungal species that may be also used fornon-industrial purposes (e.g., laboratory research). Examples ofindustrial processes may include fermentation (e.g., in production offood or beverage products), distillation, biofuel production, productionof a compound, and production of a polypeptide. Examples of industrialstrains may include, without limitation, JAY270 and ATCC4124.

In some embodiments, the fungal cell is a polyploid cell. As usedherein, a “polyploid” cell may refer to any cell whose genome is presentin more than one copy. A polyploid cell may refer to a type of cell thatis naturally found in a polyploid state, or it may refer to a cell thathas been induced to exist in a polyploid state (e.g., through specificregulation, alteration, inactivation, activation, or modification ofmeiosis, cytokinesis, or DNA replication). A polyploid cell may refer toa cell whose entire genome is polyploid, or it may refer to a cell thatis polyploid in a particular genomic locus of interest. Without wishingto be bound to theory, it is thought that the abundance of guideRNA maymore often be a rate-limiting component in genome engineering ofpolyploid cells than in haploid cells, and thus the methods using theC2c2 CRISPRS system described herein may take advantage of using acertain fungal cell type.

In some embodiments, the fungal cell is a diploid cell. As used herein,a “diploid” cell may refer to any cell whose genome is present in twocopies. A diploid cell may refer to a type of cell that is naturallyfound in a diploid state, or it may refer to a cell that has beeninduced to exist in a diploid state (e.g., through specific regulation,alteration, inactivation, activation, or modification of meiosis,cytokinesis, or DNA replication). For example, the S. cerevisiae strainS228C may be maintained in a haploid or diploid state. A diploid cellmay refer to a cell whose entire genome is diploid, or it may refer to acell that is diploid in a particular genomic locus of interest. In someembodiments, the fungal cell is a haploid cell. As used herein, a“haploid” cell may refer to any cell whose genome is present in onecopy. A haploid cell may refer to a type of cell that is naturally foundin a haploid state, or it may refer to a cell that has been induced toexist in a haploid state (e.g., through specific regulation, alteration,inactivation, activation, or modification of meiosis, cytokinesis, orDNA replication). For example, the S. cerevisiae strain S228C may bemaintained in a haploid or diploid state. A haploid cell may refer to acell whose entire genome is haploid, or it may refer to a cell that ishaploid in a particular genomic locus of interest.

As used herein, a “yeast expression vector” refers to a nucleic acidthat contains one or more sequences encoding an RNA and/or polypeptideand may further contain any desired elements that control the expressionof the nucleic acid(s), as well as any elements that enable thereplication and maintenance of the expression vector inside the yeastcell. Many suitable yeast expression vectors and features thereof areknown in the art; for example, various vectors and techniques areillustrated in in Yeast Protocols, 2nd edition, Xiao, W., ed. (HumanaPress, New York, 2007) and Buckholz, R. G. and Gleeson, M. A. (1991)Biotechnology (NY) 9(11): 1067-72. Yeast vectors may contain, withoutlimitation, a centromeric (CEN) sequence, an autonomous replicationsequence (ARS), a promoter, such as an RNA Polymerase III promoter,operably linked to a sequence or gene of interest, a terminator such asan RNA polymerase III terminator, an origin of replication, and a markergene (e.g., auxotrophic, antibiotic, or other selectable markers).Examples of expression vectors for use in yeast may include plasmids,yeast artificial chromosomes, 2μ plasmids, yeast integrative plasmids,yeast replicative plasmids, shuttle vectors, and episomal plasmids.

Stable Integration of RNA Targeting CRISP System Components in theGenome of Plants and Plant Cells

In particular embodiments, it is envisaged that the polynucleotidesencoding the components of the RNA targeting CRISPR system areintroduced for stable integration into the genome of a plant cell. Inthese embodiments, the design of the transformation vector or theexpression system can be adjusted depending on when, where and underwhat conditions the guide RNA and/or the RNA targeting gene(s) areexpressed.

In particular embodiments, it is envisaged to introduce the componentsof the RNA targeting CRISPR system stably into the genomic DNA of aplant cell. Additionally or alternatively, it is envisaged to introducethe components of the RNA targeting CRISPR system for stable integrationinto the DNA of a plant organelle such as, but not limited to a plastid,e mitochondrion or a chloroplast.

The expression system for stable integration into the genome of a plantcell may contain one or more of the following elements: a promoterelement that can be used to express the guide RNA and/or RNA targetingenzyme in a plant cell; a 5′ untranslated region to enhance expression;an intron element to further enhance expression in certain cells, suchas monocot cells; a multiple-cloning site to provide convenientrestriction sites for inserting the one or more guide RNAs and/or theRNA targeting gene sequences and other desired elements; and a 3′untranslated region to provide for efficient termination of theexpressed transcript.

The elements of the expression system may be on one or more expressionconstructs which are either circular such as a plasmid or transformationvector, or non-circular such as linear double stranded DNA.

In a particular embodiment, a RNA targeting CRISPR expression systemcomprises at least:

-   (a) a nucleotide sequence encoding a guide RNA (gRNA) that    hybridizes with a target sequence in a plant, and wherein the guide    RNA comprises a guide sequence and a direct repeat sequence, and-   (b) a nucleotide sequence encoding a RNA targeting protein,    wherein components (a) or (b) are located on the same or on    different constructs, and whereby the different nucleotide sequences    can be under control of the same or a different regulatory element    operable in a plant cell.

DNA construct(s) containing the components of the RNA targeting CRISPRsystem, and, where applicable, template sequence may be introduced intothe genome of a plant, plant part, or plant cell by a variety ofconventional techniques. The process generally comprises the steps ofselecting a suitable host cell or host tissue, introducing theconstruct(s) into the host cell or host tissue, and regenerating plantcells or plants therefrom.

In particular embodiments, the DNA construct may be introduced into theplant cell using techniques such as but not limited to electroporation,microinjection, aerosol beam injection of plant cell protoplasts, or theDNA constructs can be introduced directly to plant tissue usingbiolistic methods, such as DNA particle bombardment (see also Fu et al.,Transgenic Res. 2000 February; 9(1):11-9). The basis of particlebombardment is the acceleration of particles coated with gene/s ofinterest toward cells, resulting in the penetration of the protoplasm bythe particles and typically stable integration into the genome. (seee.g. Klein et al, Nature (1987), Klein et ah, Bio/Technology (1992),Casas et ah, Proc. Natl. Acad. Sci. USA (1993).).

In particular embodiments, the DNA constructs containing components ofthe RNA targeting CRISPR system may be introduced into the plant byAgrobacterium-mediated transformation. The DNA constructs may becombined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium lumefaciens host vector. The foreign DNA canbe incorporated into the genome of plants by infecting the plants or byincubating plant protoplasts with Agrobacterium bacteria, containing oneor more Ti (tumor-inducing) plasmids. (see e.g. Fraley et al., (1985),Rogers et al., (1987) and U.S. Pat. No. 5,563,055).

Plant Promoters

In order to ensure appropriate expression in a plant cell, thecomponents of the C2c2 CRISPR system described herein are typicallyplaced under control of a plant promoter, i.e. a promoter operable inplant cells. The use of different types of promoters is envisaged.

A constitutive plant promoter is a promoter that is able to express theopen reading frame (ORF) that it controls in all or nearly all of theplant tissues during all or nearly all developmental stages of the plant(referred to as “constitutive expression”). One non-limiting example ofa constitutive promoter is the cauliflower mosaic virus 35S promoter.The present invention envisages methods for modifying RNA sequences andas such also envisages regulating expression of plant biomolecules. Inparticular embodiments of the present invention it is thus advantageousto place one or more elements of the RNA targeting CRISPR system underthe control of a promoter that can be regulated. “Regulated promoter”refers to promoters that direct gene expression not constitutively, butin a temporally- and/or spatially-regulated manner, and includestissue-specific, tissue-preferred and inducible promoters. Differentpromoters may direct the expression of a gene in different tissues orcell types, or at different stages of development, or in response todifferent environmental conditions. In particular embodiments, one ormore of the RNA targeting CRISPR components are expressed under thecontrol of a constitutive promoter, such as the cauliflower mosaic virus35S promoter issue-preferred promoters can be utilized to targetenhanced expression in certain cell types within a particular planttissue, for instance vascular cells in leaves or roots or in specificcells of the seed. Examples of particular promoters for use in the RNAtargeting CRISPR system—are found in Kawamata et al., (1997) Plant CellPhysiol 38:792-803; Yamamoto et al., (1997) Plant J 12:255-65; Hire etal. (1992) Plant Mol Biol 20:207-18, Kuster et al, (1995) Plant Mol Biol29:759-72, and Capana et al., (1994) Plant Mol Biol 25:681-91.

Examples of promoters that are inducible and that allow forspatiotemporal control of gene editing or gene expression may use a formof energy. The form of energy may include but is not limited to soundenergy, electromagnetic radiation, chemical energy and/or thermalenergy. Examples of inducible systems include tetracycline induciblepromoters (Tet-On or Tet-Off), small molecule two-hybrid transcriptionactivations systems (FKBP, ABA, etc), or light inducible systems(Phytochrome, LOV domains, or cryptochrome), such as a Light InducibleTranscriptional Effector (LITE) that direct changes in transcriptionalactivity in a sequence-specific manner. The components of a lightinducible system may include a RNA targeting CRISPR enzyme, alight-responsive cytochrome heterodimer (e.g. from Arabidopsisthaliana), and a transcriptional activation/repression domain. Furtherexamples of inducible DNA binding proteins and methods for their use areprovided in U.S. 61/736,465 and U.S. 61/721,283, which is herebyincorporated by reference in its entirety.

In particular embodiments, transient or inducible expression can beachieved by using, for example, chemical-regulated promotors, i.e.whereby the application of an exogenous chemical induces geneexpression. Modulating of gene expression can also be obtained by achemical-repressible promoter, where application of the chemicalrepresses gene expression. Chemical-inducible promoters include, but arenot limited to, the maize ln2-2 promoter, activated by benzenesulfonamide herbicide safeners (De Veylder et al., (1997) Plant CellPhysiol 38:568-77), the maize GST promoter (GST-ll-27, WO93/01294),activated by hydrophobic electrophilic compounds used as pre-emergentherbicides, and the tobacco PR-1 a promoter (Ono et al., (2004) BiosciBiotechnol Biochem 68:803-7) activated by salicylic acid. Promoterswhich are regulated by antibiotics, such as tetracycline-inducible andtetracycline-repressible promoters (Gatz et al., (1991) Mol Gen Genet227:229-37; U.S. Pat. Nos. 5,814,618 and 5,789,156) can also be usedherein.

Translocation to and/or Expression in Specific Plant Organelles

The expression system may comprise elements for translocation to and/orexpression in a specific plant organelle.

Chloroplast Targeting

In particular embodiments, it is envisaged that the RNA targeting CRISPRsystem is used to specifically modify expression and/or translation ofchloroplast genes or to ensure expression in the chloroplast. For thispurpose use is made of chloroplast transformation methods orcompartimentalization of the RNA targeting CRISPR components to thechloroplast. For instance, the introduction of genetic modifications inthe plastid genome can reduce biosafety issues such as gene flow throughpollen.

Methods of chloroplast transformation are known in the art and includeParticle bombardment, PEG treatment, and microinjection. Additionally,methods involving the translocation of transformation cassettes from thenuclear genome to the plastid can be used as described in WO2010061186.

Alternatively, it is envisaged to target one or more of the RNAtargeting CRISPR components to the plant chloroplast. This is achievedby incorporating in the expression construct a sequence encoding achloroplast transit peptide (CTP) or plastid transit peptide, operablylinked to the 5′ region of the sequence encoding the RNA targetingprotein. The CTP is removed in a processing step during translocationinto the chloroplast. Chloroplast targeting of expressed proteins iswell known to the skilled artisan (see for instance Protein Transportinto Chloroplasts, 2010, Annual Review of Plant Biology, Vol. 61:157-180). In such embodiments it is also desired to target the one ormore guide RNAs to the plant chloroplast. Methods and constructs whichcan be used for translocating guide RNA into the chloroplast by means ofa chloroplast localization sequence are described, for instance, in US20040142476, incorporated herein by reference. Such variations ofconstructs can be incorporated into the expression systems of theinvention to efficiently translocate the RNA targeting-guide RNA(s).

Introduction of Polynucleotides Encoding the CRISPR-RNA Targeting Systemin Algal Cells.

Transgenic algae (or other plants such as rape) may be particularlyuseful in the production of vegetable oils or biofuels such as alcohols(especially methanol and ethanol) or other products. These may beengineered to express or overexpress high levels of oil or alcohols foruse in the oil or biofuel industries.

U.S. Pat. No. 8,945,839 describes a method for engineering Micro-Algae(Chlamydomonas reinhardtii cells) species) using Cas9. Using similartools, the methods of the RNA targeting CRISPR system described hereincan be applied on Chlamydomonas species and other algae. In particularembodiments, RNA targeting protein and guide RNA(s) are introduced inalgae expressed using a vector that expresses RNA targeting proteinunder the control of a constitutive promoter such as Hsp70A-Rbc S2 orBeta2-tubulin. Guide RNA is optionally delivered using a vectorcontaining T7 promoter. Alternatively, RNA targeting mRNA and in vitrotranscribed guide RNA can be delivered to algal cells. Electroporationprotocols are available to the skilled person such as the standardrecommended protocol from the GeneArt Chlamydomonas Engineering kit.

Introduction of Polynucleotides Encoding RNA Targeting Components inYeast Cells

In particular embodiments, the invention relates to the use of the RNAtargeting CRISPR system for RNA editing in yeast cells. Methods fortransforming yeast cells which can be used to introduce polynucleotidesencoding the RNA targeting CRISPR system components are well known tothe artisan and are reviewed by Kawai et al., 2010, Bioeng Bugs. 2010November-December; 1(6): 395-403). Non-limiting examples includetransformation of yeast cells by lithium acetate treatment (which mayfurther include carrier DNA and PEG treatment), bombardment or byelectroporation.

Transient Expression of RNA Targeting CRISP System Components in Plantsand Plant Cell

In particular embodiments, it is envisaged that the guide RNA and/or RNAtargeting gene are transiently expressed in the plant cell. In theseembodiments, the RNA targeting CRISPR system can ensure modification ofRNA target molecules only when both the guide RNA and the RNA targetingprotein is present in a cell, such that gene expression can further becontrolled. As the expression of the RNA targeting enzyme is transient,plants regenerated from such plant cells typically contain no foreignDNA. In particular embodiments the RNA targeting enzyme is stablyexpressed by the plant cell and the guide sequence is transientlyexpressed.

In particularly preferred embodiments, the RNA targeting CRISPR systemcomponents can be introduced in the plant cells using a plant viralvector (Scholthof et al. 1996, Annu Rev Phytopathol. 1996; 34:299-323).In further particular embodiments, said viral vector is a vector from aDNA virus. For example, geminivirus (e.g., cabbage leaf curl virus, beanyellow dwarf virus, wheat dwarf virus, tomato leaf curl virus, maizestreak virus, tobacco leaf curl virus, or tomato golden mosaic virus) ornanovirus (e.g., Faba bean necrotic yellow virus). In other particularembodiments, said viral vector is a vector from an RNA virus. Forexample, tobravirus (e.g., tobacco rattle virus, tobacco mosaic virus),potexvirus (e.g., potato virus X), or hordeivirus (e.g., barley stripemosaic virus). The replicating genomes of plant viruses arenon-integrative vectors, which is of interest in the context of avoidingthe production of GMO plants.

In particular embodiments, the vector used for transient expression ofRNA targeting CRISPR constructs is for instance a pEAQ vector, which istailored for Agrobacterium-mediated transient expression (Sainsbury F.et al., Plant Biotechnol J. 2009 September; 7(7):682-93) in theprotoplast. Precise targeting of genomic locations was demonstratedusing a modified Cabbage Leaf Curl virus (CaLCuV) vector to expressgRNAs in stable transgenic plants expressing a CRISPR enzyme (ScientificReports 5, Article number: 14926 (2015), doi:10.1038/srep14926).

In particular embodiments, double-stranded DNA fragments encoding theguide RNA and/or the RNA targeting gene can be transiently introducedinto the plant cell. In such embodiments, the introduced double-strandedDNA fragments are provided in sufficient quantity to modify RNAmolecule(s) in the cell but do not persist after a contemplated periodof time has passed or after one or more cell divisions. Methods fordirect DNA transfer in plants are known by the skilled artisan (see forinstance Davey et al. Plant Mol Biol. 1989 September; 13(3):273-85.)

In other embodiments, an RNA polynucleotide encoding the RNA targetingprotein is introduced into the plant cell, which is then translated andprocessed by the host cell generating the protein in sufficient quantityto modify the RNA molecule(s) cell (in the presence of at least oneguide RNA) but which does not persist after a contemplated period oftime has passed or after one or more cell divisions. Methods forintroducing mRNA to plant protoplasts for transient expression are knownby the skilled artisan (see for instance in Gallie, Plant Cell Reports(1993), 13; 119-122). Combinations of the different methods describedabove are also envisaged.

Delivery of RNA Targeting CRISPR Components to the Plant Cell

In particular embodiments, it is of interest to deliver one or morecomponents of the RNA targeting CRISPR system directly to the plantcell. This is of interest, inter alia, for the generation ofnon-transgenic plants (see below). In particular embodiments, one ormore of the RNA targeting components is prepared outside the plant orplant cell and delivered to the cell. For instance in particularembodiments, the RNA targeting protein is prepared in vitro prior tointroduction to the plant cell. RNA targeting protein can be prepared byvarious methods known by one of skill in the art and include recombinantproduction. After expression, the RNA targeting protein is isolated,refolded if needed, purified and optionally treated to remove anypurification tags, such as a His-tag. Once crude, partially purified, ormore completely purified RNA targeting protein is obtained, the proteinmay be introduced to the plant cell.

In particular embodiments, the RNA targeting protein is mixed with guideRNA targeting the RNA of interest to form a pre-assembledribonucleoprotein.

The individual components or pre-assembled ribonucleoprotein can beintroduced into the plant cell via electroporation, by bombardment withRNA targeting-associated gene product coated particles, by chemicaltransfection or by some other means of transport across a cell membrane.For instance, transfection of a plant protoplast with a pre-assembledCRISPR ribonucleoprotein has been demonstrated to ensure targetedmodification of the plant genome (as described by Woo et al. NatureBiotechnology, 2015; DOI: 10.1038/nbt.3389). These methods can bemodified to achieve targeted modification of RNA molecules in theplants.

In particular embodiments, the RNA targeting CRISPR system componentsare introduced into the plant cells using nanoparticles. The components,either as protein or nucleic acid or in a combination thereof, can beuploaded onto or packaged in nanoparticles and applied to the plants(such as for instance described in WO 2008042156 and US 20130185823). Inparticular, embodiments of the invention comprise nanoparticles uploadedwith or packed with DNA molecule(s) encoding the RNA targeting protein,DNA molecules encoding the guide RNA and/or isolated guide RNA asdescribed in WO2015089419.

Further means of introducing one or more components of the RNA targetingCRISPR system to the plant cell is by using cell penetrating peptides(CPP). Accordingly, in particular, embodiments the invention comprisescompositions comprising a cell penetrating peptide linked to an RNAtargeting protein. In particular embodiments of the present invention,an RNA targeting protein and/or guide RNA(s) is coupled to one or moreCPPs to effectively transport them inside plant protoplasts (Ramakrishna(2014, Genome Res. 2014 June; 24(6): 1020-7 for Cas9 in human cells). Inother embodiments, the RNA targeting gene and/or guide RNA(s) areencoded by one or more circular or non-circular DNA molecule(s) whichare coupled to one or more CPPs for plant protoplast delivery. The plantprotoplasts are then regenerated to plant cells and further to plants.CPPs are generally described as short peptides of fewer than 35 aminoacids either derived from proteins or from chimeric sequences which arecapable of transporting biomolecules across cell membrane in a receptorindependent manner. CPP can be cationic peptides, peptides havinghydrophobic sequences, amphipatic peptides, peptides having proline-richand anti-microbial sequence, and chimeric or bipartite peptides (Poogaand Langel 2005). CPPs are able to penetrate biological membranes and assuch trigger the movement of various biomolecules across cell membranesinto the cytoplasm and to improve their intracellular routing, and hencefacilitate interaction of the biolomolecule with the target. Examples ofCPP include amongst others: Tat, a nuclear transcriptional activatorprotein required for viral replication by HIV type1, penetratin, Kaposifibroblast growth factor (FGF) signal peptide sequence, integrin β3signal peptide sequence; polyarginine peptide Args sequence, Guaninerich-molecular transporters, sweet arrow peptide, etc. . . . .

Target RNA Envisaged for Plant, Algae or Fungal Applications

The target RNA, i.e. the RNA of interest, is the RNA to be targeted bythe present invention leading to the recruitment to, and the binding ofthe RNA targeting protein at, the target site of interest on the targetRNA. The target RNA may be any suitable form of RNA. This may include,in some embodiments, mRNA. In other embodiments, the target RNA mayinclude transfer RNA (tRNA) or ribosomal RNA (rRNA). In otherembodiments the target RNA may include interfering RNA (RNAi), microRNA(miRNA), microswitches, microzymes, satellite RNAs and RNA viruses. Thetarget RNA may be located in the cytoplasm of the plant cell, or in thecell nucleus or in a plant cell organelle such as a mitochondrion,chloroplast or plastid.

In particular embodiments, the RNA targeting CRISPR system is used tocleave RNA or otherwise inhibit RNA expression.

Use of RNA Targeting CRISPR System for Modulating Plant Gene ExpressionVia RNA Modulation

The RNA targeting protein may also be used, together with a suitableguide RNA, to target gene expression, via control of RNA processing. Thecontrol of RNA processing may include RNA processing reactions such asRNA splicing, including alternative splicing; viral replication (inparticular of plant viruses, including virioids in plants and tRNAbiosynthesis. The RNA targeting protein in combination with a suitableguide RNA may also be used to control RNA activation (RNAa). RNAa leadsto the promotion of gene expression, so control of gene expression maybe achieved that way through disruption or reduction of RNAa and thusless promotion of gene expression.

The RNA targeting effector protein of the invention can further be usedfor antiviral activity in plants, in particular against RNA viruses. Theeffector protein can be targeted to the viral RNA using a suitable guideRNA selective for a selected viral RNA sequence. In particular, theeffector protein may be an active nuclease that cleaves RNA, such assingle stranded RNA. provided is therefore the use of an RNA targetingeffector protein of the invention as an antiviral agent. Examples ofviruses that can be counteracted in this way include, but are notlimited to, Tobacco mosaic virus (TMV), Tomato spotted wilt virus(TSWV), Cucumber mosaic virus (CMV), Potato virus Y (PVY), Cauliflowermosaic virus (CaMV) (RT virus), Plum pox virus (PPV), Brome mosaic virus(BMV) and Potato virus X (PVX).

Examples of modulating RNA expression in plants, algae or fungi, as analternative of targeted gene modification are described herein further.

Of particular interest is the regulated control of gene expressionthrough regulated cleavage of mRNA. This can be achieved by placingelements of the RNA targeting under the control of regulated promotersas described herein.

Use of the RNA Targeting CRISPR System to Restore the Functionality oftRNA Molecules.

Pring et al describe RNA editing in plant mitochondria and chloroplaststhat alters mRNA sequences to code for different proteins than the DNA.(Plant Mol. Biol. (1993) 21 (6): 1163-1170. doi:10.1007/BF00023611). Inparticular embodiments of the invention, the elements of the RNAtargeting CRISPR system specifically targetting mitochondrial andchloroplast mRNA can be introduced in a plant or plant cell to expressdifferent proteins in such plant cell organelles mimicking the processesoccuring in vivo.

Use of the RNA Targeting CRISPR System as an Alternative to RNAInterference to Inhibit RNA Expression.

The RNA targeting CRISPR system has uses similar to RNA inhibition orRNA interference, thus can also be substituted for such methods. Inparticular embodiment, the methods of the present invention include theuse of the RNA targeting CRISPR as a substitute for e.g. an interferingribonucleic acid (such as an siRNA or shRNA or a dsRNA). Examples ofinhibition of RNA expression in plants, algae or fungi as an alternativeof targeted gene modification are described herein further.

Use of the RNA Targeting CRISPR System to Control RNA Interference.

Control over interfering RNA or miRNA may help reduce off-target effects(OTE) seen with those approaches by reducing the longevity of theinterfering RNA or miRNA in vivo or in vitro. In particular embodiments,the target RNA may include interfering RNA, i.e. RNA involved in an RNAinterference pathway, such as shRNA, siRNA and so forth. In otherembodiments, the target RNA may include microRNA (miRNA) or doublestranded RNA (dsRNA).

In other particular embodiments, if the RNA targeting protein andsuitable guide RNA(s) are selectively expressed (for example spatiallyor temporally under the control of a regulated promoter, for example atissue- or cell cycle-specific promoter and/or enhancer) this can beused to ‘protect’ the cells or systems (in vivo or in vitro) from RNAiin those cells. This may be useful in neighbouring tissues or cellswhere RNAi is not required or for the purposes of comparison of thecells or tissues where the effector protein and suitable guide are andare not expressed (i.e. where the RNAi is not controlled and where itis, respectively). The RNA targeting protein may be used to control orbind to molecules comprising or consisting of RNA, such as ribozymes,ribosomes or riboswitches. In embodiments of the invention, the guideRNA can recruit the RNA targeting protein to these molecules so that theRNA targeting protein is able to bind to them.

The RNA targeting CRISPR system of the invention can be applied in areasof in-planta RNAi technologies, without undue experimentation, from thisdisclosure, including insect pest management, plant disease managementand management of herbicide resistance, as well as in plant assay andfor other applications (see, for instance Kim et al., in PesticideBiochemistry and Physiology (Impact Factor: 2.01). January 2015; 120.DOI: 10.1016/j.pestbp.2015.01.002; Sharma et al. in Academic Journals(2015), Vol. 12(18) pp 2303-2312); Green J. M, in Pest ManagementScience, Vol 70(9), pp 1351-1357), because the present applicationprovides the foundation for informed engineering of the system.

Use of RNA Targeting CRISPR System to Modify Riboswitches and ControlMetabolic Regulation in Plants, Algae and Fungi

Riboswitches (also known as aptozymes) are regulatory segments ofmessenger RNA that bind small molecules and in turn regulate geneexpression. This mechanism allows the cell to sense the intracellularconcentration of these small molecules. A particular riboswitchtypically regulates its adjacent gene by altering the transcription, thetranslation or the splicing of this gene. Thus, in particularembodiments of the present invention, control of riboswitch activity isenvisaged through the use of the RNA targeting protein in combinationwith a suitable guide RNA to target the riboswitch. This may be throughcleavage of, or binding to, the riboswitch. In particular embodiments,reduction of riboswitch activity is envisaged. Recently, a riboswitchthat binds thiamin pyrophosphate (TPP) was characterized and found toregulate thiamin biosynthesis in plants and algae. Furthermore itappears that this element is an essential regulator of primarymetabolism in plants (Bocobza and Aharoni, Plant J. 2014 August;79(4):693-703. doi: 10.1111/tpj.12540. Epub 2014 Jun. 17). TPPriboswitches are also found in certain fungi, such as in Neurosporacrassa, where it controls alternative splicing to conditionally producean Upstream Open Reading Frame (uORF), thereby affecting the expressionof downstream genes (Cheah M T et al., (2007) Nature 447 (7143):497-500. doi:10.1038/nature05769) The RNA targeting CRISPR systemdescribed herein may be used to manipulate the endogenous riboswitchactivity in plants, algae or fungi and as such alter the expression ofdownstream genes controlled by it. In particular embodiments, the RNAtargeting CRISP system may be used in assaying riboswitch function invivo or in vitro and in studying its relevance for the metabolicnetwork. In particular embodiments the RNA targeting CRISPR system maypotentially be used for engineering of riboswitches as metabolitesensors in plants and platforms for gene control.

Use of RNA Targeting CRISPR System in RNAi Screens for Plants, Algae orFungi

Identifying gene products whose knockdown is associated with phenotypicchanges, biological pathways can be interrogated and the constituentparts identified, via RNAi screens. In particular embodiments of theinvention, control may also be exerted over or during these screens byuse of the Guide 29 or Guide 30 protein and suitable guide RNA describedherein to remove or reduce the activity of the RNAi in the screen andthus reinstate the activity of the (previously interfered with) geneproduct (by removing or reducing the interference/repression).

Use of RNA Targeting Proteins for Visualization of RNA Molecules In Vivoand In Vitro

In particular embodiments, the invention provides a nucleic acid bindingsystem. In situ hybridization of RNA with complementary probes is apowerful technique. Typically fluorescent DNA oligonucleotides are usedto detect nucleic acids by hybridization. Increased efficiency has beenattained by certain modifications, such as locked nucleic acids (LNAs),but there remains a need for efficient and versatile alternatives. Assuch, labelled elements of the RNA targeting system can be used as analternative for efficient and adaptable system for in situ hybridization

Further Applications of the RNA Targeting CRISPR System in Plants andYeasts Use of RNA Targeting CRISPR System in Biofuel Production

The term “biofuel” as used herein is an alternative fuel made from plantand plant-derived resources. Renewable biofuels can be extracted fromorganic matter whose energy has been obtained through a process ofcarbon fixation or are made through the use or conversion of biomass.This biomass can be used directly for biofuels or can be converted toconvenient energy containing substances by thermal conversion, chemicalconversion, and biochemical conversion. This biomass conversion canresult in fuel in solid, liquid, or gas form. There are two types ofbiofuels: bioethanol and biodiesel. Bioethanol is mainly produced by thesugar fermentation process of cellulose (starch), which is mostlyderived from maize and sugar cane. Biodiesel on the other hand is mainlyproduced from oil crops such as rapeseed, palm, and soybean. Biofuelsare used mainly for transportation.

Enhancing Plant Properties for Biofuel Production

In particular embodiments, the methods using the RNA targeting CRISPRsystem as described herein are used to alter the properties of the cellwall in order to facilitate access by key hydrolysing agents for a moreefficient release of sugars for fermentation. In particular embodiments,the biosynthesis of cellulose and/or lignin are modified. Cellulose isthe major component of the cell wall. The biosynthesis of cellulose andlignin are co-regulated. By reducing the proportion of lignin in a plantthe proportion of cellulose can be increased. In particular embodiments,the methods described herein are used to downregulate ligninbiosynthesis in the plant so as to increase fermentable carbohydrates.More particularly, the methods described herein are used to downregulateat least a first lignin biosynthesis gene selected from the groupconsisting of 4-coumarate 3-hydroxylase (C3H), phenylalanineammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), hydroxycinnamoyltransferase (HCT), caffeic acid O-methyltransferase (COMT), caffeoyl CoA3-O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H), cinnamylalcohol dehydrogenase (CAD), cinnamoyl CoA-reductase (CCR),4-coumarate-CoA ligase (4CL), monolignol-lignin-specificglycosyltransferase, and aldehyde dehydrogenase (ALDH) as disclosed inWO 2008064289 A2.

In particular embodiments, the methods described herein are used toproduce plant mass that produces lower levels of acetic acid duringfermentation (see also WO 2010096488).

Modifying Yeast for Biofuel Production

In particular embodiments, the RNA targeting enzyme provided herein isused for bioethanol production by recombinant micro-organisms. Forinstance, RNA targeting enzymes can be used to engineer micro-organisms,such as yeast, to generate biofuel or biopolymers from fermentablesugars and optionally to be able to degrade plant-derived lignocellulosederived from agricultural waste as a source of fermentable sugars. Moreparticularly, the invention provides methods whereby the RNA targetingCRISPR complex is used to modify the expression of endogenous genesrequired for biofuel production and/or to modify endogenous genes whymay interfere with the biofuel synthesis. More particularly the methodsinvolve stimulating the expression in a micro-organism such as a yeastof one or more nucleotide sequence encoding enzymes involved in theconversion of pyruvate to ethanol or another product of interest. Inparticular embodiments the methods ensure the stimulation of expressionof one or more enzymes which allows the micro-organism to degradecellulose, such as a cellulase. In yet further embodiments, the RNAtargeting CRISPR complex is used to suppress endogenous metabolicpathways which compete with the biofuel production pathway.

Modifying Algae and Plants for Production of Vegetable Oils or Biofuels

Transgenic algae or other plants such as rape may be particularly usefulin the production of vegetable oils or biofuels such as alcohols(especially methanol and ethanol), for instance. These may be engineeredto express or overexpress high levels of oil or alcohols for use in theoil or biofuel industries.

U.S. Pat. No. 8,945,839 describes a method for engineering Micro-Algae(Chlamydomonas reinhardtii cells) species) using Cas9. Using similartools, the methods of the RNA targeting CRISPR system described hereincan be applied on Chlamydomonas species and other algae. In particularembodiments, the RNA targeting effetor protein and guide RNA areintroduced in algae expressed using a vector that expresses the RNAtargeting effector protein under the control of a constitutive promotersuch as Hsp70A-Rbc S2 or Beta2-tubulin. Guide RNA will be deliveredusing a vector containing T7 promoter. Alternatively, in vitrotranscribed guide RNA can be delivered to algae cells. Electroporationprotocol follows standard recommended protocol from the GeneArtChlamydomonas Engineering kit.

Particular Applications of the RNA Targeting Enzymes in Plants

In particular embodiments, present invention can be used as a therapyfor virus removal in plant systems as it is able to cleave viral RNA.Previous studies in human systems have demonstrated the success ofutilizing CRISPR in targeting the single strand RNA virus, hepatitis C(A. Price, et al., Proc. Natl. Acad. Sci, 2015). These methods may alsobe adapted for using the RNA targeting CRISPR system in plants.

Improved Plants

The present invention also provides plants and yeast cells obtainableand obtained by the methods provided herein. The improved plantsobtained by the methods described herein may be useful in food or feedproduction through the modified expression of genes which, for instanceensure tolerance to plant pests, herbicides, drought, low or hightemperatures, excessive water, etc.

The improved plants obtained by the methods described herein, especiallycrops and algae may be useful in food or feed production throughexpression of, for instance, higher protein, carbohydrate, nutrient orvitamin levels than would normally be seen in the wildtype. In thisregard, improved plants, especially pulses and tubers are preferred.

Improved algae or other plants such as rape may be particularly usefulin the production of vegetable oils or biofuels such as alcohols(especially methanol and ethanol), for instance. These may be engineeredto express or overexpress high levels of oil or alcohols for use in theoil or biofuel industries.

The invention also provides for improved parts of a plant. Plant partsinclude, but are not limited to, leaves, stems, roots, tubers, seeds,endosperm, ovule, and pollen. Plant parts as envisaged herein may beviable, nonviable, regeneratable, and/or non-regeneratable.

It is also encompassed herein to provide plant cells and plantsgenerated according to the methods of the invention. Gametes, seeds,embryos, either zygotic or somatic, progeny or hybrids of plantscomprising the genetic modification, which are produced by traditionalbreeding methods, are also included within the scope of the presentinvention. Such plants may contain a heterologous or foreign DNAsequence inserted at or instead of a target sequence. Alternatively,such plants may contain only an alteration (mutation, deletion,insertion, substitution) in one or more nucleotides. As such, suchplants will only be different from their progenitor plants by thepresence of the particular modification.

In an embodiment of the invention, a C2c2 system is used to engineerpathogen resistant plants, for example by creating resistance againstdiseases caused by bacteria, fungi or viruses. In certain embodiments,pathogen resistance can be accomplished by engineering crops to producea C2c2 system that wil be ingested by an insect pest, leading tomortality. In an embodiment of the invention, a C2c2 system is used toengineer abiotic stress tolerance. In another embodiment, a C2c2 systemis used to engineer drought stress tolerance or salt stress tolerance,or cold or heat stress tolerance. Younis et al. 2014, Int. J. Biol. Sci.10; 1150 reviewed potential targets of plant breeding methods, all ofwhich are amenable to correction or improvement through use of a C2c2system described herein. Some non-limiting target crops includeArabidops Zea mays is thaliana, Oryza sativa L, Prunus domestica L.,Gossypium hirsutum, Nicotiana rustica, Zea mays, Medicago sativa,Nicotiana benthamiana and Arabidopsis thaliana

In an embodiment of the invention, a C2c2 system is used for managementof crop pests. For example, a C2c2 system operable in a crop pest can beexpressed from a plant host or transferred directly to the target, forexample using a viral vector.

In an embodiment, the invention provides a method of efficientlyproducing homozygous organisms from a heterozygous non-human startingorganism. In an embodiment, the invention is used in plant breeding. Inanother embodiment, the invention is used in animal breeding. In suchembodiments, a homozygous organism such as a plant or animal is made bypreventing or suppressing recombination by interfering with at least onetarget gene involved in double strand breaks, chromosome pairing and/orstrand exchange.

Application of the C2C2 Proteins in Optimized Functional RNA TargetingSystems

In an aspect the invention provides a system for specific delivery offunctional components to the RNA environment. This can be ensured usingthe CRISPR systems comprising the RNA targeting effector proteins of thepresent invention which allow specific targeting of different componentsto RNA. More particularly such components include activators orrepressors, such as activators or repressors of RNA translation,degradation, etc. Applications of this system are described elsewhereherein.

According to one aspect the invention provides non-naturally occurringor engineered composition comprising a guide RNA comprising a guidesequence capable of hybridizing to a target sequence in a genomic locusof interest in a cell, wherein the guide RNA is modified by theinsertion of one or more distinct RNA sequence(s) that bind an adaptorprotein. In particular embodiments, the RNA sequences may bind to two ormore adaptor proteins (e.g. aptamers), and wherein each adaptor proteinis associated with one or more functional domains. The guide RNAs of thec2c2 enzymes described herein are shown to be amenable to modificationof the guide sequence. In particular embodiments, the guide RNA ismodified by the insertion of distinct RNA sequence(s) 5′ of the directrepeat, within the direct repeat, or 3′ of the guide sequence. Whenthere is more than one functional domain, the functional domains can besame or different, e.g., two of the same or two different activators orrepressors. In an aspect the invention provides a herein-discussedcomposition, wherein the one or more functional domains are attached tothe RNA targeting enzyme so that upon binding to the target RNA thefunctional domain is in a spatial orientation allowing for thefunctional domain to function in its attributed function; In an aspectthe invention provides a herein-discussed composition, wherein thecomposition comprises a CRISPR-Cas complex having at least threefunctional domains, at least one of which is associated with the RNAtargeting enzyme and at least two of which are associated with the gRNA.

Accordingly, In an aspect the invention provides non-naturally occurringor engineered CRISPR-Cas complex composition comprising the guide RNA asherein-discussed and a CRISPR enzyme which is an RNA targeting enzyme,wherein optionally the RNA targeting enzyme comprises at least onemutation, such that the RNA targeting enzyme has no more than 5% of thenuclease activity of the enzyme not having the at least one mutation,and optionally one or more comprising at least one or more nuclearlocalization sequences. In particular embodiments, the guide RNA isadditionally or alternatively modified so as to still ensure binding ofthe RNA targeting enzyme but to prevent cleavage by the RNA targetingenzyme (as detailed elsewhere herein).

In particular embodiments, the RNA targeting enzyme is a c2c2 enzymewhich has a diminished nuclease activity of at least 97%, or 100% ascompared with the c2c2 enzyme not having the at least one mutation. Inan aspect the invention provides a herein-discussed composition, whereinthe C2c2 enzyme comprises two or more mutations. The mutations may beselected from mutations of one or more of the following amino acidresidues: R597, H602, R1278, and H1283, such as for instance one or moreof the following mutations: R597A, H602A, R1278A, and H1283A, accordingto Leptotrichia shahii c2c2 protein or a corresponding position in anortholog.

In particular embodiments, an RNA targeting system is provided asdescribed herein above comprising two or more functional domains. Inparticular embodiments, the two or more functional domains areheterologous functional domain. In particular embodiments, the systemcomprises an adaptor protein which is a fusion protein comprising afunctional domain, the fusion protein optionally comprising a linkerbetween the adaptor protein and the functional domain. In particularembodiments, the linker includes a GlySer linker. Additionally oralternatively, one or more functional domains are attached to the RNAeffector protein by way of a linker, optionally a GlySer linker. Inparticular embodiments, the one or more functional domains are attachedto the RNA targeting enzyme through one or both of the HEPN domains.

In an aspect the invention provides a herein-discussed composition,wherein the one or more functional domains associated with the adaptorprotein or the RNA targeting enzume is a domain capable of activating orrepressing RNA translation. In an aspect the invention provides aherein-discussed composition, wherein at least one of the one or morefunctional domains associated with the adaptor protein have one or moreactivities comprising methylase activity, demethylase activity,transcription activation activity, transcription repression activity,transcription release factor activity, histone modification activity,DNA integration activity RNA cleavage activity, DNA cleavage activity ornucleic acid binding activity, or molecular switch activity or chemicalinducibility or light inducibility.

In an aspect the invention provides a herein-discussed compositioncomprising an aptamer sequence. In particular embodiments, the aptamersequence is two or more aptamer sequences specific to the same adaptorprotein. In an aspect the invention provides a herein-discussedcomposition, wherein the aptamer sequence is two or more aptamersequences specific to different adaptor protein. In an aspect theinvention provides a herein-discussed composition, wherein the adaptorprotein comprises MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34,JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5,ϕCb8r, ϕCb12r, ϕCb23r, 7s, PRR1. Accordingly, in particular embodiments,the aptamer is selected from a binding protein specifically binding anyone of the adaptor proteins listed above. In an aspect the inventionprovides a herein-discussed composition, wherein the cell is aeukaryotic cell. In an aspect the invention provides a herein-discussedcomposition, wherein the eukaryotic cell is a mammalian cell, a plantcell or a yeast cell, whereby the mammalian cell is optionally a mousecell. In an aspect the invention provides a herein-discussedcomposition, wherein the mammalian cell is a human cell.

In an aspect the invention provides a herein above-discussed compositionwherein there is more than one gRNA, and the gRNAs target differentsequences whereby when the composition is employed, there ismultiplexing. In an aspect the invention provides a composition whereinthere is more than one gRNA modified by the insertion of distinct RNAsequence(s) that bind to one or more adaptor proteins.

In an aspect the invention provides a herein-discussed compositionwherein one or more adaptor proteins associated with one or morefunctional domains is present and bound to the distinct RNA sequence(s)inserted into the guide RNA(s).

In an aspect the invention provides a herein-discussed compositionwherein the guide RNA is modified to have at least one non-codingfunctional loop; e.g., wherein the at least one non-coding functionalloop is repressive; for instance, wherein at least one non-codingfunctional loop comprises Alu.

In an aspect the invention provides a method for modifying geneexpression comprising the administration to a host or expression in ahost in vivo of one or more of the compositions as herein-discussed.

In an aspect the invention provides a herein-discussed method comprisingthe delivery of the composition or nucleic acid molecule(s) codingtherefor, wherein said nucleic acid molecule(s) are operatively linkedto regulatory sequence(s) and expressed in vivo. In an aspect theinvention provides a herein-discussed method wherein the expression invivo is via a lentivirus, an adenovirus, or an AAV.

In an aspect the invention provides a mammalian cell line of cells asherein-discussed, wherein the cell line is, optionally, a human cellline or a mouse cell line. In an aspect the invention provides atransgenic mammalian model, optionally a mouse, wherein the model hasbeen transformed with a herein-discussed composition or is a progeny ofsaid transformant.

In an aspect the invention provides a nucleic acid molecule(s) encodingguide RNA or the RNA targeting CRISPR-Cas complex or the composition asherein-discussed. In an aspect the invention provides a vectorcomprising: a nucleic acid molecule encoding a guide RNA (gRNA)comprising a guide sequence capable of hybridizing to a target sequencein a genomic locus of interest in a cell, wherein the direct repeat ofthe gRNA is modified by the insertion of distinct RNA sequence(s) thatbind(s) to two or more adaptor proteins, and wherein each adaptorprotein is associated with one or more functional domains; or, whereinthe gRNA is modified to have at least one non-coding functional loop. Inan aspect the invention provides vector(s) comprising nucleic acidmolecule(s) encoding: non-naturally occurring or engineered CRISPR-Cascomplex composition comprising the gRNA herein-discussed, and an RNAtargeting enzyme, wherein optionally the RNA targeting enzyme comprisesat least one mutation, such that the RNA targeting enzyme has no morethan 5% of the nuclease activity of the RNA targeting enzyme not havingthe at least one mutation, and optionally one or more comprising atleast one or more nuclear localization sequences. In an aspect a vectorcan further comprise regulatory element(s) operable in a eukaryotic celloperably linked to the nucleic acid molecule encoding the guide RNA(gRNA) and/or the nucleic acid molecule encoding the RNA targetingenzyme and/or the optional nuclear localization sequence(s).

In one aspect, the invention provides a kit comprising one or more ofthe components described hereinabove. In some embodiments, the kitcomprises a vector system as described above and instructions for usingthe kit.

In an aspect the invention provides a method of screening for gain offunction (GOF) or loss of function (LOF) or for screening non-codingRNAs or potential regulatory regions (e.g. enhancers, repressors)comprising the cell line of as herein-discussed or cells of the modelherein-discussed containing or expressing the RNA targeting enzyme andintroducing a composition as herein-discussed into cells of the cellline or model, whereby the gRNA includes either an activator or arepressor, and monitoring for GOF or LOF respectively as to those cellsas to which the introduced gRNA includes an activator or as to thosecells as to which the introduced gRNA includes a repressor.

In an aspect the invention provides a library of non-naturally occurringor engineered compositions, each comprising a RNA targeting CRISPR guideRNA (gRNA) comprising a guide sequence capable of hybridizing to atarget RNA sequence of interest in a cell, an RNA targeting enzyme,wherein the RNA targeting enzyme comprises at least one mutation, suchthat the RNA targeting enzyme has no more than 5% of the nucleaseactivity of the RNA targeting enzyme not having the at least onemutation, wherein the gRNA is modified by the insertion of distinct RNAsequence(s) that bind to one or more adaptor proteins, and wherein theadaptor protein is associated with one or more functional domains,wherein the composition comprises one or more or two or more adaptorproteins, wherein the each protein is associated with one or morefunctional domains, and wherein the gRNAs comprise a genome wide librarycomprising a plurality of RNA targeting guide RNAs (gRNAs). In an aspectthe invention provides a library as herein-discussed, wherein the RNAtargeting RNA targeting enzyme has a diminished nuclease activity of atleast 97%, or 100% as compare with the RNA targeting enzyme not havingthe at least one mutation. In an aspect the invention provides a libraryas herein-discussed, wherein the adaptor protein is a fusion proteincomprising the functional domain. In an aspect the invention provides alibrary as herein discussed, wherein the gRNA is not modified by theinsertion of distinct RNA sequence(s) that bind to the one or two ormore adaptor proteins. In an aspect the invention provides a library asherein discussed, wherein the one or two or more functional domains areassociated with the RNA targeting enzyme. In an aspect the inventionprovides a library as herein discussed, wherein the cell population ofcells is a population of eukaryotic cells. In an aspect the inventionprovides a library as herein discussed, wherein the eukaryotic cell is amammalian cell, a plant cell or a yeast cell. In an aspect the inventionprovides a library as herein discussed, wherein the mammalian cell is ahuman cell. In an aspect the invention provides a library as hereindiscussed, wherein the population of cells is a population of embryonicstem (ES) cells.

In an aspect the invention provides a library as herein discussed,wherein the targeting is of about 100 or more RNA sequences. In anaspect the invention provides a library as herein discussed, wherein thetargeting is of about 1000 or more RNA sequences. In an aspect theinvention provides a library as herein discussed, wherein the targetingis of about 20,000 or more sequences. In an aspect the inventionprovides a library as herein discussed, wherein the targeting is of theentire transcriptome. In an aspect the invention provides a library asherein discussed, wherein the targeting is of a panel of targetsequences focused on a relevant or desirable pathway. In an aspect theinvention provides a library as herein discussed, wherein the pathway isan immune pathway. In an aspect the invention provides a library asherein discussed, wherein the pathway is a cell division pathway.

In one aspect, the invention provides a method of generating a modeleukaryotic cell comprising a gene with modified expression. In someembodiments, a disease gene is any gene associated an increase in therisk of having or developing a disease. In some embodiments, the methodcomprises (a) introducing one or more vectors encoding the components ofthe system described herein above into a eukaryotic cell, and (b)allowing a CRISPR complex to bind to a target polynucleotide so as tomodify expression of a gene, thereby generating a model eukaryotic cellcomprising modified gene expression.

The structural information provided herein allows for interrogation ofguide RNA interaction with the target RNA and the RNA targeting enzymepermitting engineering or alteration of guide RNA structure to optimizefunctionality of the entire RNA targeting CRISPR-Cas system. Forexample, the guide RNA may be extended, without colliding with the RNAtargeting protein by the insertion of adaptor proteins that can bind toRNA. These adaptor proteins can further recruit effector proteins orfusions which comprise one or more functional domains.

An aspect of the invention is that the above elements are comprised in asingle composition or comprised in individual compositions. Thesecompositions may advantageously be applied to a host to elicit afunctional effect on the genomic level.

The skilled person will understand that modifications to the guide RNAwhich allow for binding of the adapter+functional domain but not properpositioning of the adapter+functional domain (e.g. due to sterichindrance within the three dimensial structure of the CRISPR complex)are modifications which are not intended. The one or more modified guideRNA may be modified, by introduction of a distinct RNA sequence(s) 5′ ofthe direct repeat, within the direct repeat, or 3′ of the guidesequence.

The modified guide RNA, the inactivated RNA targeting enzyme (with orwithout functional domains), and the binding protein with one or morefunctional domains, may each individually be comprised in a compositionand administered to a host individually or collectively. Alternatively,these components may be provided in a single composition foradministration to a host. Administration to a host may be performed viaviral vectors known to the skilled person or described herein fordelivery to a host (e.g. lentiviral vector, adenoviral vector, AAVvector). As explained herein, use of different selection markers (e.g.for lentiviral gRNA selection) and concentration of gRNA (e.g. dependenton whether multiple gRNAs are used) may be advantageous for eliciting animproved effect.

Using the provided compositions, the person skilled in the art canadvantageously and specifically target single or multiple loci with thesame or different functional domains to elicit one or more genomicevents. The compositions may be applied in a wide variety of methods forscreening in libraries in cells and functional modeling in vivo (e.g.gene activation of lincRNA and indentification of function;gain-of-function modeling; loss-of-function modeling; the use thecompositions of the invention to establish cell lines and transgenicanimals for optimization and screening purposes).

The current invention comprehends the use of the compositions of thecurrent invention to establish and utilize conditional or inducibleCRISPR RNA targeting events. (See, e.g., Platt et al., Cell (2014),http://dx.doi.org/10.1016/j.cell.2014.09.014, or PCT patent publicationscited herein, such as WO 2014/093622 (PCT/US2013/074667), which are notbelieved prior to the present invention or application). For example,the target cell comprises RNA targeting CRISRP enzyme conditionally orinducibly (e.g. in the form of Cre dependent constructs) and/or theadapter protein conditionally or inducibly and, on expression of avector introduced into the target cell, the vector expresses that whichinduces or gives rise to the condition of s RNA targeting enzymeexpression and/or adaptor expression in the target cell. By applying theteaching and compositions of the current invention with the known methodof creating a CRISPR complex, inducible gene expression affected byfunctional domains are also an aspect of the current invention.Alternatively, the adaptor protein may be provided as a conditional orinducible element with a conditional or inducible s RNA targeting enzymeto provide an effective model for screening purposes, whichadvantageously only requires minimal design and administration ofspecific gRNAs for a broad number of applications.

Guide RNA According to the Invention Comprising a Dead Guide Sequence

In one aspect, the invention provides guide sequences which are modifiedin a manner which allows for formation of the CRISPR complex andsuccessful binding to the target, while at the same time, not allowingfor successful nuclease activity (i.e. without nuclease activity/withoutindel activity). For matters of explanation such modified guidesequences are referred to as “dead guides” or “dead guide sequences”.These dead guides or dead guide sequences can be thought of ascatalytically inactive or conformationally inactive with regard tonuclease activity. Indeed, dead guide sequences may not sufficientlyengage in productive base pairing with respect to the ability to promotecatalytic activity or to distinguish on-target and off-target bindingactivity. Briefly, the assay involves synthesizing a CRISPR target RNAand guide RNAs comprising mismatches with the target RNA, combiningthese with the RNA targeting enzyme and analyzing cleavage based on gelsbased on the presence of bands generated by cleavage products, andquantifying cleavage based upon relative band intensities.

Hence, in a related aspect, the invention provides a non-naturallyoccurring or engineered composition RNA targeting CRISPR-Cas systemcomprising a functional RNA targeting as described herein, and guide RNA(gRNA) wherein the gRNA comprises a dead guide sequence whereby the gRNAis capable of hybridizing to a target sequence such that the RNAtargeting CRISPR-Cas system is directed to a genomic locus of interestin a cell without detectable RNA cleavage activity of a non-mutant RNAtargeting enzyme of the system. It is to be understood that any of thegRNAs according to the invention as described herein elsewhere may beused as dead gRNAs/gRNAs comprising a dead guide sequence as describedherein below. Any of the methods, products, compositions and uses asdescribed herein elsewhere is equally applicable with the deadgRNAs/gRNAs comprising a dead guide sequence as further detailed below.By means of further guidance, the following particular aspects andembodiments are provided.

The ability of a dead guide sequence to direct sequence-specific bindingof a CRISPR complex to an RNA target sequence may be assessed by anysuitable assay. For example, the components of a CRISPR systemsufficient to form a CRISPR complex, including the dead guide sequenceto be tested, may be provided to a host cell having the correspondingtarget sequence, such as by transfection with vectors encoding thecomponents of the CRISPR sequence, followed by an assessment ofpreferential cleavage within the target sequence. For instance, cleavageof a target RNA polynucleotide sequence may be evaluated in a test tubeby providing the target sequence, components of a CRISPR complex,including the dead guide sequence to be tested and a control guidesequence different from the test dead guide sequence, and comparingbinding or rate of cleavage at the target sequence between the test andcontrol guide sequence reactions. Other assays are possible, and willoccur to those skilled in the art. A dead guide sequence may be selectedto target any target sequence. In some embodiments, the target sequenceis a sequence within a genome of a cell.

As explained further herein, several structural parameters allow for aproper framework to arrive at such dead guides. Dead guide sequences aretypically shorter than respective guide sequences which result in activeRNA cleavage. In particular embodiments, dead guides are 5%, 10%, 20%,30%, 40%, 50%, shorter than respective guides directed to the same.

As explained below and known in the art, one aspect of gRNA—RNAtargeting specificity is the direct repeat sequence, which is to beappropriately linked to such guides. In particular, this implies thatthe direct repeat sequences are designed dependent on the origin of theRNA targeting enzyme. Thus, structural data available for validated deadguide sequences may be used for designing C2c2 specific equivalents.Structural similarity between, e.g., the orthologous nuclease domainsHEPN of two or more C2c2 effector proteins may be used to transferdesign equivalent dead guides. Thus, the dead guide herein may beappropriately modified in length and sequence to reflect such C2c2specific equivalents, allowing for formation of the CRISPR complex andsuccessful binding to the target RNA, while at the same time, notallowing for successful nuclease activity.

The use of dead guides in the context herein as well as the state of theart provides a surprising and unexpected platform for network biologyand/or systems biology in both in vitro, ex vivo, and in vivoapplications, allowing for multiplex gene targeting, and in particularbidirectional multiplex gene targeting. Prior to the use of dead guides,addressing multiple targets has been challenging and in some cases notpossible. With the use of dead guides, multiple targets, and thusmultiple activities, may be addressed, for example, in the same cell, inthe same animal, or in the same patient. Such multiplexing may occur atthe same time or staggered for a desired timeframe.

For example, the dead guides allow to use gRNA as a means for genetargeting, without the consequence of nuclease activity, while at thesame time providing directed means for activation or repression. GuideRNA comprising a dead guide may be modified to further include elementsin a manner which allow for activation or repression of gene activity,in particular protein adaptors (e.g. aptamers) as described hereinelsewhere allowing for functional placement of gene effectors (e.g.activators or repressors of gene activity). One example is theincorporation of aptamers, as explained herein and in the state of theart. By engineering the gRNA comprising a dead guide to incorporateprotein-interacting aptamers (Konermann et al., “Genome-scaletranscription activation by an engineered CRISPR-Cas9 complex,”doi:10.1038/nature14136, incorporated herein by reference), one mayassemble multiple distinct effector domains. Such may be modeled afternatural processes.

Thus, one aspect is a gRNA of the invention which comprises a deadguide, wherein the gRNA further comprises modifications which providefor gene activation or repression, as described herein. The dead gRNAmay comprise one or more aptamers. The aptamers may be specific to geneeffectors, gene activators or gene repressors. Alternatively, theaptamers may be specific to a protein which in turn is specific to andrecruits/binds a specific gene effector, gene activator or generepressor. If there are multiple sites for activator or repressorrecruitment, it is preferred that the sites are specific to eitheractivators or repressors. If there are multiple sites for activator orrepressor binding, the sites may be specific to the same activators orsame repressors. The sites may also be specific to different activatorsor different repressors. The effectors, activators, repressors may bepresent in the form of fusion proteins.

In an aspect, the invention provides a method of selecting a dead guideRNA targeting sequence for directing a functionalized CRISPR system to agene locus in an organism, which comprises: a) locating one or moreCRISPR motifs in the gene locus; b) analyzing the 20 nt sequencedownstream of each CRISPR motif by: i) determining the GC content of thesequence; and ii) determining whether there are off-target matches ofthe first 15 nt of the sequence in the genome of the organism; c)selecting the sequence for use in a guide RNA if the GC content of thesequence is 70% or less and no off-target matches are identified. In anembodiment, the sequence is selected if the GC content is 50% or less.In an embodiment, the sequence is selected if the GC content is 40% orless. In an embodiment, the sequence is selected if the GC content is30% or less. In an embodiment, two or more sequences are analyzed andthe sequence having the lowest GC content is selected. In an embodiment,off-target matches are determined in regulatory sequences of theorganism. In an embodiment, the gene locus is a regulatory region. Anaspect provides a dead guide RNA comprising the targeting sequenceselected according to the aforementioned methods.

In an aspect, the invention provides a dead guide RNA for targeting afunctionalized CRISPR system to a gene locus in an organism. In anembodiment of the invention, the dead guide RNA comprises a targetingsequence wherein the CG content of the target sequence is 70% or less,and the first 15 nt of the targeting sequence does not match anoff-target sequence downstream from a CRISPR motif in the regulatorysequence of another gene locus in the organism. In certain embodiments,the GC content of the targeting sequence 60% or less, 55% or less, 50%or less, 45% or less, 40% or less, 35% or less or 30% or less. Incertain embodiments, the GC content of the targeting sequence is from70% to 60% or from 60% to 50% or from 50% to 40% or from 40% to 30%. Inan embodiment, the targeting sequence has the lowest CG content amongpotential targeting sequences of the locus.

In an embodiment of the invention, the first 15 nt of the dead guidematch the target sequence. In another embodiment, first 14 nt of thedead guide match the target sequence. In another embodiment, the first13 nt of the dead guide match the target sequence. In another embodimentfirst 12 nt of the dead guide match the target sequence. In anotherembodiment, first 11 nt of the dead guide match the target sequence. Inanother embodiment, the first 10 nt of the dead guide match the targetsequence. In an embodiment of the invention the first 15 nt of the deadguide does not match an off-target sequence downstream from a CRISPRmotif in the regulatory region of another gene locus. In otherembodiments, the first 14 nt, or the first 13 nt of the dead guide, orthe first 12 nt of the guide, or the first 11 nt of the dead guide, orthe first 10 nt of the dead guide, does not match an off-target sequencedownstream from a CRISPR motif in the regulatory region of another genelocus. In other embodiments, the first 15 nt, or 14 nt, or 13 nt, or 12nt, or 11 nt of the dead guide do not match an off-target sequencedownstream from a CRISPR motif in the genome.

In certain embodiments, the dead guide RNA includes additionalnucleotides at the 3′-end that do not match the target sequence. Thus, adead guide RNA that includes the first 20-28 nt, downstream of a CRISPRmotif can be extended in length at the 3′ end.

General Provisions

In an aspect, the invention provides a nucleic acid binding system. Insitu hybridization of RNA with complementary probes is a powerfultechnique. Typically fluorescent DNA oligonucleotides are used to detectnucleic acids by hybridization. Increased efficiency has been attainedby certain modifications, such as locked nucleic acids (LNAs), but thereremains a need for efficient and versatile alternatives. The inventionprovides an efficient and adaptable system for in situ hybridization.

In embodiments of the invention the terms guide sequence and guide RNAare used interchangeably as in foregoing cited documents such as WO2014/093622 (PCT/US2013/074667). In general, a guide sequence is anypolynucleotide sequence having sufficient complementarity with a targetpolynucleotide sequence to hybridize with the target sequence and directsequence-specific binding of a CRISPR complex to the target sequence. Insome embodiments, the degree of complementarity between a guide sequenceand its corresponding target sequence, when optimally aligned using asuitable alignment algorithm, is about or more than about 50%, 60%, 75%,80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may bedetermined with the use of any suitable algorithm for aligningsequences, non-limiting example of which include the Smith-Watermanalgorithm, the Needleman-Wunsch algorithm, algorithms based on theBurrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW,Clustal X, BLAT, Novoalign (Novocraft Technologies; available atwww.novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (availableat soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). Insome embodiments, a guide sequence is about or more than about 5, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In someembodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30,25, 20, 15, 12, or fewer nucleotides in length. Preferably the guidesequence is 10-30 nucleotides long. The ability of a guide sequence todirect sequence-specific binding of a CRISPR complex to a targetsequence may be assessed by any suitable assay. For example, thecomponents of a CRISPR system sufficient to form a CRISPR complex,including the guide sequence to be tested, may be provided to a hostcell having the corresponding target sequence, such as by transfectionwith vectors encoding the components of the CRISPR sequence, followed byan assessment of preferential cleavage within the target sequence, suchas by Surveyor assay as described herein. Similarly, cleavage of atarget polynucleotide sequence may be evaluated in a test tube byproviding the target sequence, components of a CRISPR complex, includingthe guide sequence to be tested and a control guide sequence differentfrom the test guide sequence, and comparing binding or rate of cleavageat the target sequence between the test and control guide sequencereactions. Other assays are possible, and will occur to those skilled inthe art. A guide sequence may be selected to target any target sequence.In some embodiments, the target sequence is a sequence within a genomeof a cell. Exemplary target sequences include those that are unique inthe target genome.

In general, and throughout this specification, the term “vector” refersto a nucleic acid molecule capable of transporting another nucleic acidto which it has been linked. Vectors include, but are not limited to,nucleic acid molecules that are single-stranded, double-stranded, orpartially double-stranded; nucleic acid molecules that comprise one ormore free ends, no free ends (e.g., circular); nucleic acid moleculesthat comprise DNA, RNA, or both; and other varieties of polynucleotidesknown in the art. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments canbe inserted, such as by standard molecular cloning techniques. Anothertype of vector is a viral vector, wherein virally-derived DNA or RNAsequences are present in the vector for packaging into a virus (e.g.,retroviruses, replication defective retroviruses, adenoviruses,replication defective adenoviruses, and adeno-associated viruses). Viralvectors also include polynucleotides carried by a virus for transfectioninto a host cell. Certain vectors are capable of autonomous replicationin a host cell into which they are introduced (e.g., bacterial vectorshaving a bacterial origin of replication and episomal mammalianvectors). Other vectors (e.g., non-episomal mammalian vectors) areintegrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively-linked. Such vectors are referred toherein as “expression vectors.” Vectors for and that result inexpression in a eukaryotic cell can be referred to herein as “eukaryoticexpression vectors.” Common expression vectors of utility in recombinantDNA techniques are often in the form of plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.,in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell).

The term “regulatory element” is intended to include promoters,enhancers, internal ribosomal entry sites (IRES), and other expressioncontrol elements (e.g., transcription termination signals, such aspolyadenylation signals and poly-U sequences). Such regulatory elementsare described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).Regulatory elements include those that direct constitutive expression ofa nucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). A tissue-specific promoter maydirect expression primarily in a desired tissue of interest, such asmuscle, neuron, bone, skin, blood, specific organs (e.g., liver,pancreas), or particular cell types (e.g., lymphocytes). Regulatoryelements may also direct expression in a temporal-dependent manner, suchas in a cell-cycle dependent or developmental stage-dependent manner,which may or may not also be tissue or cell-type specific. In someembodiments, a vector comprises one or more pol III promoter (e.g., 1,2, 3, 4, 5, or more pol III promoters), one or more pol II promoters(e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol Ipromoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), orcombinations thereof. Examples of pol III promoters include, but are notlimited to, U6 and H1 promoters. Examples of pol II promoters include,but are not limited to, the retroviral Rous sarcoma virus (RSV) LTRpromoter (optionally with the RSV enhancer), the cytomegalovirus (CMV)promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al,Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductasepromoter, the f3-actin promoter, the phosphoglycerol kinase (PGK)promoter, and the EF1α promoter. Also encompassed by the term“regulatory element” are enhancer elements, such as WPRE; CMV enhancers;the R-U5′ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p.466-472, 1988); SV40 enhancer; and the intron sequence between exons 2and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p.1527-31, 1981). It will be appreciated by those skilled in the art thatthe design of the expression vector can depend on such factors as thechoice of the host cell to be transformed, the level of expressiondesired, etc. A vector can be introduced into host cells to therebyproduce transcripts, proteins, or peptides, including fusion proteins orpeptides, encoded by nucleic acids as described herein (e.g., clusteredregularly interspersed short palindromic repeats (CRISPR) transcripts,proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).

Advantageous vectors include lentiviruses and adeno-associated viruses,and types of such vectors can also be selected for targeting particulartypes of cells.

As used herein, the term “crRNA” or “guide RNA” or “single guide RNA” or“sgRNA” or “one or more nucleic acid components” of a Type V or Type VICRISPR-Cas locus effector protein comprises any polynucleotide sequencehaving sufficient complementarity with a target nucleic acid sequence tohybridize with the target nucleic acid sequence and directsequence-specific binding of a nucleic acid-targeting complex to thetarget nucleic acid sequence. In some embodiments, the degree ofcomplementarity, when optimally aligned using a suitable alignmentalgorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%,95%, 97.5%, 99%, or more. Optimal alignment may be determined with theuse of any suitable algorithm for aligning sequences, non-limitingexample of which include the Smith-Waterman algorithm, theNeedleman-Wunsch algorithm, algorithms based on the Burrows-WheelerTransform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X,BLAT, Novoalign (Novocraft Technologies; available atwww.novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (availableat soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).The ability of a guide sequence (within a nucleic acid-targeting guideRNA) to direct sequence-specific binding of a nucleic acid-targetingcomplex to a target nucleic acid sequence may be assessed by anysuitable assay. For example, the components of a nucleic acid-targetingCRISPR system sufficient to form a nucleic acid-targeting complex,including the guide sequence to be tested, may be provided to a hostcell having the corresponding target nucleic acid sequence, such as bytransfection with vectors encoding the components of the nucleicacid-targeting complex, followed by an assessment of preferentialtargeting (e.g., cleavage) within the target nucleic acid sequence, suchas by Surveyor assay as described herein. Similarly, cleavage of atarget nucleic acid sequence may be evaluated in a test tube byproviding the target nucleic acid sequence, components of a nucleicacid-targeting complex, including the guide sequence to be tested and acontrol guide sequence different from the test guide sequence, andcomparing binding or rate of cleavage at the target sequence between thetest and control guide sequence reactions. Other assays are possible,and will occur to those skilled in the art. A guide sequence, and hencea nucleic acid-targeting guide RNA may be selected to target any targetnucleic acid sequence. The target sequence may be DNA. The targetsequence may be any RNA sequence. In some embodiments, the targetsequence may be a sequence within a RNA molecule selected from the groupconsisting of messenger RNA (mRNA), pre-mRNA, ribosomaal RNA (rRNA),transfer RNA (tRNA), micro-RNA (miRNA), small interfering RNA (siRNA),small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), double strandedRNA (dsRNA), non coding RNA (ncRNA), long non-coding RNA (lncRNA), andsmall cytoplasmatic RNA (scRNA). In some preferred embodiments, thetarget sequence may be a sequence within a RNA molecule selected fromthe group consisting of mRNA, pre-mRNA, and rRNA. In some preferredembodiments, the target sequence may be a sequence within a RNA moleculeselected from the group consisting of ncRNA, and lncRNA. In some morepreferred embodiments, the target sequence may be a sequence within anmRNA molecule or a pre-mRNA molecule.

In some embodiments, a nucleic acid-targeting guide RNA is selected toreduce the degree secondary structure within the RNA-targeting guideRNA. In some embodiments, about or less than about 75%, 50%, 40%, 30%,25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the nucleicacid-targeting guide RNA participate in self-complementary base pairingwhen optimally folded. Optimal folding may be determined by any suitablepolynucleotide folding algorithm. Some programs are based on calculatingthe minimal Gibbs free energy. An example of one such algorithm ismFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981),133-148). Another example folding algorithm is the online webserverRNAfold, developed at Institute for Theoretical Chemistry at theUniversity of Vienna, using the centroid structure prediction algorithm(see e.g., A. R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carrand GM Church, 2009, Nature Biotechnology 27(12): 1151-62).

In certain embodiments, a guide RNA or crRNA may comprise, consistessentially of, or consist of a direct repeat (DR) sequence and a guidesequence or spacer sequence. In certain embodiments, the guide RNA orcrRNA may comprise, consist essentially of, or consist of a directrepeat sequence fused or linked to a guide sequence or spacer sequence.In certain embodiments, the direct repeat sequence may be locatedupstream (i.e., 5′) from the guide sequence or spacer sequence. In otherembodiments, the direct repeat sequence may be located downstream (i.e.,3′) from the guide sequence or spacer sequence.

In certain embodiments, the crRNA comprises a stem loop, preferably asingle stem loop. In certain embodiments, the direct repeat sequenceforms a stem loop, preferably a single stem loop.

In certain embodiments, the spacer length of the guide RNA is from 15 to35 nt. In certain embodiments, the spacer length of the guide RNA is atleast 15 nucleotides, preferably at least 18 nt, such at at least 19,20, 21, 22, or more nt. In certain embodiments, the spacer length isfrom 15 to 17 nt, e.g., 15, 16, or 17 nt, from 17 to 20 nt, e.g., 17,18, 19, or 20 nt, from 20 to 24 nt, e.g., 20, 21, 22, 23, or 24 nt, from23 to 25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt, e.g., 24, 25, 26,or 27 nt, from 27-30 nt, e.g., 27, 28, 29, or 30 nt, from 30-35 nt,e.g., 30, 31, 32, 33, 34, or 35 nt, or 35 nt or longer.

Applicants also perform a challenge experiment to verify the DNAtargeting and cleaving capability of a Type V protein such as C2c1 orC2c3. This experiment closely parallels similar work in E coli for theheterologous expression of StCas9 (Sapranauskas, R. et al. Nucleic AcidsRes 39, 9275-9282 (2011)). Applicants introduce a plasmid containingboth a PAM and a resistance gene into the heterologous E. coli, and thenplate on the corresponding antibiotic. If there is DNA cleavage of theplasmid, Applicants observe no viable colonies.

In further detail, the assay is as follows for a DNA target. Two E. colistrains are used in this assay. One carries a plasmid that encodes theendogenous effector protein locus from the bacterial strain. The otherstrain carries an empty plasmid (e.g. pACYC184, control strain). Allpossible 7 or 8 bp PAM sequences are presented on an antibioticresistance plasmid (pUC19 with ampicillin resistance gene). The PAM islocated next to the sequence of proto-spacer 1 (the DNA target to thefirst spacer in the endogenous effector protein locus). Two PAMlibraries were cloned. One has a 8 random bp 5′ of the proto-spacer(e.g. total of 65536 different PAM sequences=complexity). The otherlibrary has 7 random bp 3′ of the proto-spacer (e.g. total complexity is16384 different PAMs). Both libraries were cloned to have in average 500plasmids per possible PAM. Test strain and control strain weretransformed with 5′PAM and 3′PAM library in separate transformations andtransformed cells were plated separately on ampicillin plates.Recognition and subsequent cutting/interference with the plasmid rendersa cell vulnerable to ampicillin and prevents growth. Approximately 12 hafter transformation, all colonies formed by the test and controlstrains where harvested and plasmid DNA was isolated. Plasmid DNA wasused as template for PCR amplification and subsequent deep sequencing.Representation of all PAMs in the untransfomed libraries showed theexpected representation of PAMs in transformed cells. Representation ofall PAMs found in control strains showed the actual representation.Representation of all PAMs in test strain showed which PAMs are notrecognized by the enzyme and comparison to the control strain allowsextracting the sequence of the depleted PAM.

For minimization of toxicity and off-target effect, it will be importantto control the concentration of nucleic acid-targeting guide RNAdelivered. Optimal concentrations of nucleic acid-targeting guide RNAcan be determined by testing different concentrations in a cellular ornon-human eukaryote animal model and using deep sequencing the analyzethe extent of modification at potential off-target genomic loci. Theconcentration that gives the highest level of on-target modificationwhile minimizing the level of off-target modification should be chosenfor in vivo delivery. The nucleic acid-targeting system is derivedadvantageously from a Type VI CRISPR system. In some embodiments, one ormore elements of a nucleic acid-targeting system is derived from aparticular organism comprising an endogenous RNA-targeting system. Inparticular embodiments, the Type VI RNA-targeting Cas enzyme is C2c2.Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3,Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12),Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3,Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17,Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4,homologues thereof, or modified versions thereof. In embodiments, theType VI protein such as C2c2as referred to herein also encompasses ahomologue or an orthologue of a Type VI protein such as C2c2. The terms“orthologue” (also referred to as “ortholog” herein) and “homologue”(also referred to as “homolog” herein) are well known in the art. Bymeans of further guidance, a “homologue” of a protein as used herein isa protein of the same species which performs the same or a similarfunction as the protein it is a homologue of. Homologous proteins maybut need not be structurally related, or are only partially structurallyrelated. An “orthologue” of a protein as used herein is a protein of adifferent species which performs the same or a similar function as theprotein it is an orthologue of. Orthologous proteins may but need not bestructurally related, or are only partially structurally related. Inparticular embodiments, the homologue or orthologue of a Type VI proteinsuch as C2c2as referred to herein has a sequence homology or identity ofat least 80%, more preferably at least 85%, even more preferably atleast 90%, such as for instance at least 95% with a Type VI protein suchas C2c2. In further embodiments, the homologue or orthologue of a TypeVI protein such as C2c2as referred to herein has a sequence identity ofat least 80%, more preferably at least 85%, even more preferably atleast 90%, such as for instance at least 95% with the wild type Type VIprotein such as C2c2.

In an embodiment, the Type VI RNA-targeting Cas protein may be aC2c2ortholog of an organism of a genus which includes but is not limitedto Corynebacter, Sutterella, Legionella, Treponema, Filifactor,Eubacterium, Streptococcus, Lactobacillus, Mycoplasma; Bacteroides,Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum,Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus,Nitratifractor, Mycoplasma and Campylobacter. Species of organism ofsuch a genus can be as otherwise herein discussed.

Some methods of identifying orthologs of CRISPR-Cas system enzymes mayinvolve identifying tracr sequences in genomes of interest.Identification of tracr sequences may relate to the following steps:Search for the direct repeats or tract mate sequences in a database toidentify a CRISPR region comprising a CRISPR enzyme. Search forhomologous sequences in the CRISPR region flanking the CRISPR enzyme inboth the sense and antisense directions. Look for transcriptionalterminators and secondary structures. Identify any sequence that is nota direct repeat or a tract mate sequence but has more than 50% identityto the direct repeat or tract mate sequence as a potential tracrsequence. Take the potential tracr sequence and analyze fortranscriptional terminator sequences associated therewith.

It will be appreciated that any of the functionalities described hereinmay be engineered into CRISPR enzymes from other orthologs, includingchimeric enzymes comprising fragments from multiple orthologs. Examplesof such orthologs are described elsewhere herein. Thus, chimeric enzymesmay comprise fragments of CRISPR enzyme orthologs of an organism whichincludes but is not limited to Corynebacter, Sutterella, Legionella,Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus,Mycoplasma Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta,Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum,Staphylococcus, Nitratifractor, Mycoplasma and Campylobacter. A chimericenzyme can comprise a first fragment and a second fragment, and thefragments can be of CRISPR enzyme orthologs of organisms of genusesherein mentioned or of species herein mentioned; advantageously thefragments are from CRISPR enzyme orthologs of different species.

In embodiments, the Type VI RNA-targeting effector protein, inparticular the C2c2 protein as referred to herein also encompasses afunctional variant of C2c2 or a homologue or an orthologue thereof. A“functional variant” of a protein as used herein refers to a variant ofsuch protein which retains at least partially the activity of thatprotein. Functional variants may include mutants (which may beinsertion, deletion, or replacement mutants), including polymorphs, etc.Also included within functional variants are fusion products of suchprotein with another, usually unrelated, nucleic acid, protein,polypeptide or peptide. Functional variants may be naturally occurringor may be man-made. Advantageous embodiments can involve engineered ornon-naturally occurring Type VI RNA-targeting effector protein, e.g.,C2c1/C2c3 or an ortholog or homolog thereof.

In an embodiment, nucleic acid molecule(s) encoding the Type VIRNA-targeting effector protein, in particular C2c2 or an ortholog orhomolog thereof, may be codon-optimized for expression in an eukaryoticcell. A eukaryote can be as herein discussed. Nucleic acid molecule(s)can be engineered or non-naturally occurring.

In an embodiment, the Type VI RNA-targeting effector protein, inparticular C2c2 or an ortholog or homolog thereof, may comprise one ormore mutations (and hence nucleic acid molecule(s) coding for same mayhave mutation(s). The mutations may be artificially introduced mutationsand may include but are not limited to one or more mutations in acatalytic domain. Examples of catalytic domains with reference to a Cas9enzyme may include but are not limited to RuvC I, RuvC II, RuvC III andHNH domains.

In an embodiment, the Type VI protein such as C2c2 or an ortholog orhomolog thereof, may comprise one or more mutations. The mutations maybe artificially introduced mutations and may include but are not limitedto one or more mutations in a catalytic domain. Examples of catalyticdomains with reference to a Cas enzyme may include but are not limitedto RuvC I, RuvC II, RuvC III, HNH domains, and HEPN domains.

In an embodiment, the Type VI protein such as C2c2 or an ortholog orhomolog thereof, may be used as a generic nucleic acid binding proteinwith fusion to or being operably linked to a functional domain.Exemplary functional domains may include but are not limited totranslational initiator, translational activator, translationalrepressor, nucleases, in particular ribonucleases, a spliceosome, beads,a light inducible/controllable domain or a chemicallyinducible/controllable domain.

In some embodiments, the unmodified nucleic acid-targeting effectorprotein may have cleavage activity. In some embodiments, theRNA-targeting effector protein may direct cleavage of one or bothnucleic acid (DNA or RNA) strands at the location of or near a targetsequence, such as within the target sequence and/or within thecomplement of the target sequence or at sequences associated with thetarget sequence. In some embodiments, the nucleic acid-targeting Casprotein may direct cleavage of one or both DNA or RNA strands withinabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, ormore base pairs from the first or last nucleotide of a target sequence.In some embodiments, the cleavage may be blunt, i.e., generating bluntends. In some embodiments, the cleavage may be staggered, i.e.,generating sticky ends. In some embodiments, the cleavage may be astaggered cut with a 5′ overhang, e.g., a 5′ overhang of 1 to 5nucleotides. In some embodiments, the cleavage may be a staggered cutwith a 3′ overhang, e.g., a 3′ overhang of 1 to 5 nucleotides. In someembodiments, a vector encodes a nucleic acid-targeting Cas protein thatmay be mutated with respect to a corresponding wild-type enzyme suchthat the mutated nucleic acid-targeting Cas protein lacks the ability tocleave one or both DNA or RNA strands of a target polynucleotidecontaining a target sequence. As a further example, two or morecatalytic domains of Cas (RuvC I, RuvC II, and RuvC III or the HNHdomain, or HEPN domain) may be mutated to produce a mutated Cassubstantially lacking all RNA cleavage activity. As described herein,corresponding catalytic domains of a C2c2 effector protein may also bemutated to produce a mutated C2c2 effector protein lacking all DNAcleavage activity or having substantially reduced DNA cleavage activity.In some embodiments, a nucleic acid-targeting effector protein may beconsidered to substantially lack all RNA cleavage activity when the RNAcleavage activity of the mutated enzyme is about no more than 25%, 10%,5%, 1%, 0.1%, 0.01%, or less of the nucleic acid cleavage activity ofthe non-mutated form of the enzyme; an example can be when the nucleicacid cleavage activity of the mutated form is nil or negligible ascompared with the non-mutated form. An effector protein may beidentified with reference to the general class of enzymes that sharehomology to the biggest nuclease with multiple nuclease domains from theType V/Type VI CRISPR system. Most preferably, the effector protein is aType V/Type VI protein such as C2c2. By derived, Applicants mean thatthe derived enzyme is largely based, in the sense of having a highdegree of sequence homology with, a wildtype enzyme, but that it hasbeen mutated (modified) in some way as known in the art or as describedherein.

Again, it will be appreciated that the terms Cas and CRISPR enzyme andCRISPR protein and Cas protein are generally used interchangeably and atall points of reference herein refer by analogy to novel CRISPR effectorproteins further described in this application, unless otherwiseapparent, such as by specific reference to Cas9. As mentioned above,many of the residue numberings used herein refer to the effector proteinfrom the Type V/Type VI CRISPR locus. However, it will be appreciatedthat this invention includes many more effector proteins from otherspecies of microbes. In certain embodiments, Cas may be constitutivelypresent or inducibly present or conditionally present or administered ordelivered. Cas optimization may be used to enhance function or todevelop new functions, one can generate chimeric Cas proteins. And Casmay be used as a generic nucleic acid binding protein.

Typically, in the context of an endogenous nucleic acid-targetingsystem, formation of a nucleic acid-targeting complex (comprising aguide RNA hybridized to a target sequence and complexed with one or morenucleic acid-targeting effector proteins) results in cleavage of one orboth DNA or RNA strands in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 50, or more base pairs from) the target sequence. As usedherein the term “sequence(s) associated with a target locus of interest”refers to sequences near the vicinity of the target sequence (e.g.within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs fromthe target sequence, wherein the target sequence is comprised within atarget locus of interest).

An example of a codon optimized sequence, is in this instance a sequenceoptimized for expression in a eukaryote, e.g., humans (i.e. beingoptimized for expression in humans), or for another eukaryote, animal ormammal as herein discussed; see, e.g., SaCas9 human codon optimizedsequence in WO 2014/093622 (PCT/US2013/074667) as an example of a codonoptimized sequence (from knowledge in the art and this disclosure, codonoptimizing coding nucleic acid molecule(s), especially as to effectorprotein (e.g., C2c2) is within the ambit of the skilled artisan). Whilstthis is preferred, it will be appreciated that other examples arepossible and codon optimization for a host species other than human, orfor codon optimization for specific organs is known. In someembodiments, an enzyme coding sequence encoding a DNA/RNA-targeting Casprotein is codon optimized for expression in particular cells, such aseukaryotic cells. The eukaryotic cells may be those of or derived from aparticular organism, such as a mammal, including but not limited tohuman, or non-human eukaryote or animal or mammal as herein discussed,e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal orprimate. In some embodiments, processes for modifying the germ linegenetic identity of human beings and/or processes for modifying thegenetic identity of animals which are likely to cause them sufferingwithout any substantial medical benefit to man or animal, and alsoanimals resulting from such processes, may be excluded. In general,codon optimization refers to a process of modifying a nucleic acidsequence for enhanced expression in the host cells of interest byreplacing at least one codon (e.g., about or more than about 1, 2, 3, 4,5, 10, 15, 20, 25, 50, or more codons) of the native sequence withcodons that are more frequently or most frequently used in the genes ofthat host cell while maintaining the native amino acid sequence. Variousspecies exhibit particular bias for certain codons of a particular aminoacid. Codon bias (differences in codon usage between organisms) oftencorrelates with the efficiency of translation of messenger RNA (mRNA),which is in turn believed to be dependent on, among other things, theproperties of the codons being translated and the availability ofparticular transfer RNA (tRNA) molecules. The predominance of selectedtRNAs in a cell is generally a reflection of the codons used mostfrequently in peptide synthesis. Accordingly, genes can be tailored foroptimal gene expression in a given organism based on codon optimization.Codon usage tables are readily available, for example, at the “CodonUsage Database” available at www.kazusa.orjp/codon/and these tables canbe adapted in a number of ways. See Nakamura, Y., et al. “Codon usagetabulated from the international DNA sequence databases: status for theyear 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codonoptimizing a particular sequence for expression in a particular hostcell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), arealso available. In some embodiments, one or more codons (e.g., 1, 2, 3,4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encodinga DNA/RNA-targeting Cas protein corresponds to the most frequently usedcodon for a particular amino acid.

In some embodiments, a vector encodes a nucleic acid-targeting effectorprotein such as the Type V RNA-targeting effector protein, in particularC2c2, or an ortholog or homolog thereof comprising one or more nuclearlocalization sequences (NLSs), such as about or more than about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, theRNA-targeting effector protein comprises about or more than about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus,about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs ator near the carboxy-terminus, or a combination of these (e.g., zero orat least one or more NLS at the amino-terminus and zero or at one ormore NLS at the carboxy terminus). When more than one NLS is present,each may be selected independently of the others, such that a single NLSmay be present in more than one copy and/or in combination with one ormore other NLSs present in one or more copies. In some embodiments, anNLS is considered near the N- or C-terminus when the nearest amino acidof the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, ormore amino acids along the polypeptide chain from the N- or C-terminus.Non-limiting examples of NLSs include an NLS sequence derived from: theNLS of the SV40 virus large T-antigen, having the amino acid sequencePKKKRKV; the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartiteNLS with the sequence KRPAATKKAGQAKKKK); the c-myc NLS having the aminoacid sequence PAAKRVKLD or RQRRNELKRSP; the hRNPA1 M9 NLS having thesequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; the sequenceRMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV of the IBB domain fromimportin-alpha; the sequences VSRKRPRP and PPKKARED of the myoma Tprotein; the sequence POPKKKPL of human p53; the sequence SALIKKKKKMAPof mouse c-abl IV; the sequences DRLRR and PKQKKRK of the influenzavirus NS1; the sequence RKLKKKIKKL of the Hepatitis virus delta antigen;the sequence REKKKFLKRR of the mouse Mx1 protein; the sequenceKRKGDEVDGVDEVAKKKSKK of the human poly(ADP-ribose) polymerase; and thesequence RKCLQAGMNLEARKTKK of the steroid hormone receptors (human)glucocorticoid. In general, the one or more NLSs are of sufficientstrength to drive accumulation of the DNA/RNA-targeting Cas protein in adetectable amount in the nucleus of a eukaryotic cell. In general,strength of nuclear localization activity may derive from the number ofNLSs in the nucleic acid-targeting effector protein, the particularNLS(s) used, or a combination of these factors. Detection ofaccumulation in the nucleus may be performed by any suitable technique.For example, a detectable marker may be fused to the nucleicacid-targeting protein, such that location within a cell may bevisualized, such as in combination with a means for detecting thelocation of the nucleus (e.g., a stain specific for the nucleus such asDAPI). Cell nuclei may also be isolated from cells, the contents ofwhich may then be analyzed by any suitable process for detectingprotein, such as immunohistochemistry, Western blot, or enzyme activityassay. Accumulation in the nucleus may also be determined indirectly,such as by an assay for the effect of nucleic acid-targeting complexformation (e.g., assay for DNA or RNA cleavage or mutation at the targetsequence, or assay for altered gene expression activity affected by DNAor RNA-targeting complex formation and/or DNA or RNA-targeting Casprotein activity), as compared to a control not exposed to the nucleicacid-targeting Cas protein or nucleic acid-targeting complex, or exposedto a nucleic acid-targeting Cas protein lacking the one or more NLSs. Inpreferred embodiments of the herein described C2c2 effector proteincomplexes and systems the codon optimized C2c2 effector proteinscomprise an NLS attached to the C-terminal of the protein.

In some embodiments, one or more vectors driving expression of one ormore elements of a nucleic acid-targeting system are introduced into ahost cell such that expression of the elements of the nucleicacid-targeting system direct formation of a nucleic acid-targetingcomplex at one or more target sites. For example, a nucleicacid-targeting effector enzyme and a nucleic acid-targeting guide RNAcould each be operably linked to separate regulatory elements onseparate vectors. RNA(s) of the nucleic acid-targeting system can bedelivered to a transgenic nucleic acid-targeting effector protein animalor mammal, e.g., an animal or mammal that constitutively or inducibly orconditionally expresses nucleic acid-targeting effector protein; or ananimal or mammal that is otherwise expressing nucleic acid-targetingeffector protein or has cells containing nucleic acid-targeting effectorprotein, such as by way of prior administration thereto of a vector orvectors that code for and express in vivo nucleic acid-targetingeffector protein. Alternatively, two or more of the elements expressedfrom the same or different regulatory elements, may be combined in asingle vector, with one or more additional vectors providing anycomponents of the nucleic acid-targeting system not included in thefirst vector. nucleic acid-targeting system elements that are combinedin a single vector may be arranged in any suitable orientation, such asone element located 5′ with respect to (“upstream” of) or 3′ withrespect to (“downstream” of) a second element. The coding sequence ofone element may be located on the same or opposite strand of the codingsequence of a second element, and oriented in the same or oppositedirection. In some embodiments, a single promoter drives expression of atranscript encoding a nucleic acid-targeting effector protein and thenucleic acid-targeting guide RNA, embedded within one or more intronsequences (e.g., each in a different intron, two or more in at least oneintron, or all in a single intron). In some embodiments, the nucleicacid-targeting effector protein and the nucleic acid-targeting guide RNAmay be operably linked to and expressed from the same promoter. Deliveryvehicles, vectors, particles, nanoparticles, formulations and componentsthereof for expression of one or more elements of a nucleicacid-targeting system are as used in the foregoing documents, such as WO2014/093622 (PCT/US2013/074667). In some embodiments, a vector comprisesone or more insertion sites, such as a restriction endonucleaserecognition sequence (also referred to as a “cloning site”). In someembodiments, one or more insertion sites (e.g., about or more than about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are locatedupstream and/or downstream of one or more sequence elements of one ormore vectors. In some embodiments, a vector comprises two or moreinsertion sites, so as to allow insertion of a guide sequence at eachsite. In such an arrangement, the two or more guide sequences maycomprise two or more copies of a single guide sequence, two or moredifferent guide sequences, or combinations of these. When multipledifferent guide sequences are used, a single expression construct may beused to target nucleic acid-targeting activity to multiple different,corresponding target sequences within a cell. For example, a singlevector may comprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, or more guide sequences. In some embodiments, about or morethan about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more suchguide-sequence-containing vectors may be provided, and optionallydelivered to a cell. In some embodiments, a vector comprises aregulatory element operably linked to an enzyme-coding sequence encodinga a nucleic acid-targeting effector protein. nucleic acid-targetingeffector protein or nucleic acid-targeting guide RNA or RNA(s) can bedelivered separately; and advantageously at least one of these isdelivered via a particle or nanoparticle complex. nucleic acid-targetingeffector protein mRNA can be delivered prior to the nucleicacid-targeting guide RNA to give time for nucleic acid-targetingeffector protein to be expressed. nucleic acid-targeting effectorprotein mRNA might be administered 1-12 hours (preferably around 2-6hours) prior to the administration of nucleic acid-targeting guide RNA.Alternatively, nucleic acid-targeting effector protein mRNA and nucleicacid-targeting guide RNA can be administered together. Advantageously, asecond booster dose of guide RNA can be administered 1-12 hours(preferably around 2-6 hours) after the initial administration ofnucleic acid-targeting effector protein mRNA+guide RNA. Additionaladministrations of nucleic acid-targeting effector protein mRNA and/orguide RNA might be useful to achieve the most efficient levels of genomeand/or transcriptome modification.

In one aspect, the invention provides methods for using one or moreelements of a nucleic acid-targeting system. The nucleic acid-targetingcomplex of the invention provides an effective means for modifying atarget DNA or RNA single or double stranded, linear or super-coiled).The nucleic acid-targeting complex of the invention has a wide varietyof utility including modifying (e.g., deleting, inserting,translocating, inactivating, activating) a target DNA or RNA in amultiplicity of cell types. As such the nucleic acid-targeting complexof the invention has a broad spectrum of applications in, e.g., genetherapy, drug screening, disease diagnosis, and prognosis. An exemplarynucleic acid-targeting complex comprises a DNA or RNA-targeting effectorprotein complexed with a guide RNA hybridized to a target sequencewithin the target locus of interest.

In one embodiment, this invention provides a method of cleaving a targetRNA. The method may comprise modifying a target RNA using a nucleicacid-targeting complex that binds to the target RNA and effect cleavageof said target RNA. In an embodiment, the nucleic acid-targeting complexof the invention, when introduced into a cell, may create a break (e.g.,a single or a double strand break) in the RNA sequence. For example, themethod can be used to cleave a disease RNA in a cell For example, anexogenous RNA template comprising a sequence to be integrated flanked byan upstream sequence and a downstream sequence may be introduced into acell. The upstream and downstream sequences share sequence similaritywith either side of the site of integration in the RNA. Where desired, adonor RNA can be mRNA. The exogenous RNA template comprises a sequenceto be integrated (e.g., a mutated RNA). The sequence for integration maybe a sequence endogenous or exogenous to the cell. Examples of asequence to be integrated include RNA encoding a protein or a non-codingRNA (e.g., a microRNA). Thus, the sequence for integration may beoperably linked to an appropriate control sequence or sequences.Alternatively, the sequence to be integrated may provide a regulatoryfunction. The upstream and downstream sequences in the exogenous RNAtemplate are selected to promote recombination between the RNA sequenceof interest and the donor RNA. The upstream sequence is a RNA sequencethat shares sequence similarity with the RNA sequence upstream of thetargeted site for integration. Similarly, the downstream sequence is aRNA sequence that shares sequence similarity with the RNA sequencedownstream of the targeted site of integration. The upstream anddownstream sequences in the exogenous RNA template can have 75%, 80%,85%, 90%, 95%, or 100% sequence identity with the targeted RNA sequence.Preferably, the upstream and downstream sequences in the exogenous RNAtemplate have about 95%, 96%, 97%, 98%, 99%, or 100% sequence identitywith the targeted RNA sequence. In some methods, the upstream anddownstream sequences in the exogenous RNA template have about 99% or100% sequence identity with the targeted RNA sequence. An upstream ordownstream sequence may comprise from about 20 bp to about 2500 bp, forexample, about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200,2300, 2400, or 2500 bp. In some methods, the exemplary upstream ordownstream sequence have about 200 bp to about 2000 bp, about 600 bp toabout 1000 bp, or more particularly about 700 bp to about 1000 bp. Insome methods, the exogenous RNA template may further comprise a marker.Such a marker may make it easy to screen for targeted integrations.Examples of suitable markers include restriction sites, fluorescentproteins, or selectable markers. The exogenous RNA template of theinvention can be constructed using recombinant techniques (see, forexample, Sambrook et al., 2001 and Ausubel et al., 1996). In a methodfor modifying a target RNA by integrating an exogenous RNA template, abreak (e.g., double or single stranded break in double or singlestranded DNA or RNA) is introduced into the DNA or RNA sequence by thenucleic acid-targeting complex, the break is repaired via homologousrecombination with an exogenous RNA template such that the template isintegrated into the RNA target. The presence of a double-stranded breakfacilitates integration of the template. In other embodiments, thisinvention provides a method of modifying expression of a RNA in aeukaryotic cell. The method comprises increasing or decreasingexpression of a target polynucleotide by using a nucleic acid-targetingcomplex that binds to the DNA or RNA (e.g., mRNA or pre-mRNA). In somemethods, a target RNA can be inactivated to effect the modification ofthe expression in a cell. For example, upon the binding of aRNA-targeting complex to a target sequence in a cell, the target RNA isinactivated such that the sequence is not translated, the coded proteinis not produced, or the sequence does not function as the wild-typesequence does. For example, a protein or microRNA coding sequence may beinactivated such that the protein or microRNA or pre-microRNA transcriptis not produced. The target RNA of a RNA-targeting complex can be anyRNA endogenous or exogenous to the eukaryotic cell. For example, thetarget RNA can be a RNA residing in the nucleus of the eukaryotic cell.The target RNA can be a sequence (e.g., mRNA or pre-mRNA) coding a geneproduct (e.g., a protein) or a non-coding sequence (e.g., ncRNA, lncRNA,tRNA, or rRNA). Examples of target RNA include a sequence associatedwith a signaling biochemical pathway, e.g., a signaling biochemicalpathway-associated RNA. Examples of target RNA include a diseaseassociated RNA. A “disease-associated” RNA refers to any RNA which isyielding translation products at an abnormal level or in an abnormalform in cells derived from a disease-affected tissues compared withtissues or cells of a non disease control. It may be a RNA transcribedfrom a gene that becomes expressed at an abnormally high level; it maybe a RNA transcribed from a gene that becomes expressed at an abnormallylow level, where the altered expression correlates with the occurrenceand/or progression of the disease. A disease-associated RNA also refersto a RNA transcribed from a gene possessing mutation(s) or geneticvariation that is directly responsible or is in linkage disequilibriumwith a gene(s) that is responsible for the etiology of a disease. Thetranslated products may be known or unknown, and may be at a normal orabnormal level. The target RNA of a RNA-targeting complex can be any RNAendogenous or exogenous to the eukaryotic cell. For example, the targetRNA can be a RNA residing in the nucleus of the eukaryotic cell. Thetarget RNA can be a sequence (e.g., mRNA or pre-mRNA) coding a geneproduct (e.g., a protein) or a non-coding sequence (e.g., ncRNA, lncRNA,tRNA, or rRNA).

In some embodiments, the method may comprise allowing a nucleicacid-targeting complex to bind to the target DNA or RNA to effectcleavage of said target DNA or RNA thereby modifying the target DNA orRNA, wherein the nucleic acid-targeting complex comprises a nucleicacid-targeting effector protein complexed with a guide RNA hybridized toa target sequence within said target DNA or RNA. In one aspect, theinvention provides a method of modifying expression of DNA or RNA in aeukaryotic cell. In some embodiments, the method comprises allowing anucleic acid-targeting complex to bind to the DNA or RNA such that saidbinding results in increased or decreased expression of said DNA or RNA;wherein the nucleic acid-targeting complex comprises a nucleicacid-targeting effector protein complexed with a guide RNA. Similarconsiderations and conditions apply as above for methods of modifying atarget DNA or RNA. In fact, these sampling, culturing andre-introduction options apply across the aspects of the presentinvention. In one aspect, the invention provides for methods ofmodifying a target DNA or RNA in a eukaryotic cell, which may be invivo, ex vivo or in vitro. In some embodiments, the method comprisessampling a cell or population of cells from a human or non-human animal,and modifying the cell or cells. Culturing may occur at any stage exvivo. The cell or cells may even be re-introduced into the non-humananimal or plant. For re-introduced cells it is particularly preferredthat the cells are stem cells.

Indeed, in any aspect of the invention, the nucleic acid-targetingcomplex may comprise a nucleic acid-targeting effector protein complexedwith a guide RNA hybridized to a target sequence.

The invention relates to the engineering and optimization of systems,methods and compositions used for the control of gene expressioninvolving DNA or RNA sequence targeting, that relate to the nucleicacid-targeting system and components thereof. In advantageousembodiments, the effector proteinenzyme is a Type VI protein such asC2c2. An advantage of the present methods is that the CRISPR systemminimizes or avoids off-target binding and its resulting side effects.This is achieved using systems arranged to have a high degree ofsequence specificity for the target DNA or RNA.

In relation to a nucleic acid-targeting complex or system preferably,the tracr sequence has one or more hairpins and is 30 or morenucleotides in length, 40 or more nucleotides in length, or 50 or morenucleotides in length; the crRNA sequence is between 10 to 30nucleotides in length, the nucleic acid-targeting effector protein is aType VI effector protein.

In certain embodiments, the effector protein may be a Listeria sp.C2c2p, preferably Listeria seeligeria C2c2p, more preferably Listeriaseeligeria serovar ½b str. SLCC3954 C2c2p and the crRNA sequence may be44 to 47 nucleotides in length, with a 5′ 29-nt direct repeat (DR) and a15-nt to 18-nt spacer.

In certain embodiments, the effector protein may be a Leptotrichia sp.C2c2p, preferably Leptotrichia shahii C2c2p, more preferablyLeptotrichia shahii DSM 19757 C2c2p and the crRNA sequence may be 42 to58 nucleotides in length, with a 5′direct repeat of at least 24 nt, suchas a 5′ 24-28-nt direct repeat (DR) and a spacer of at least 14 nt, suchas a 14-nt to 28-nt spacer, or a spacer of at least 18 nt, such as 19,20, 21, 22, or more nt, such as 18-28, 19-28, 20-28, 21-28, or 22-28 nt.

In certain embodiments, the effector protein may be a Type VI locieffector protein, more particularly a C2c2p, and the crRNA sequence maybe 36 to 63 nucleotides in length, preferably 37-nt to 62-nt in length,or 38-nt to 61-nt in length, or 39-nt to 60-nt in length, morepreferably 40-nt to 59-nt in length, or 41-nt to 58-nt in length, mostpreferably 42-nt to 57-nt in length. For example, the crRNA maycomprise, consist essentially of or consist of a direct repeat (DR),preferably a 5′ DR, 26-nt to 31-nt in length, preferably 27-nt to 30-ntin length, even more preferably 28-nt or 29-nt in length or at least 28or 29 nt in length, and a spacer 10-nt to 32-nt in length, preferably11-nt to 31-nt in length, more preferably 12-nt to 30-nt in length, evenmore preferably 13-nt to 29-nt in length, and most preferably 14-nt to28-nt in length, such as 18-28 nt, 19-28 nt, 20-28 nt, 21-28 nt, or22-28 nt.

In certain embodiments, the effector protein may be a Type VI locieffector protein, more particularly a C2c2p, and the tracrRNA sequencemay be at least 60-nt long, such as at least 65-nt in length, or atleast 70-nt in length, such as from 60-nt to 70-nt in length, or from60-nt to 70-nt in length, or from 70-nt to 80-nt in length, or from80-nt to 90-nt in length, or from 90-nt to 100-nt in length, or from100-nt to 110-nt in length, or from 110-nt to 120-nt in length, or from120-nt to 130-nt in length, or from 130-nt to 140-nt in length, or from140-nt to 150-nt in length, or more than 150-nt in length. Seeillustrative examples in FIG. 22-37.

In certain embodiments, the effector protein may be a Type VI locieffector protein, more particularly a C2c2p, and no tracrRNA may berequired for cleavage.

The use of two different aptamers (each associated with a distinctnucleic acid-targeting guide RNAs) allows an activator-adaptor proteinfusion and a repressor-adaptor protein fusion to be used, with differentnucleic acid-targeting guide RNAs, to activate expression of one DNA orRNA, whilst repressing another. They, along with their different guideRNAs can be administered together, or substantially together, in amultiplexed approach. A large number of such modified nucleicacid-targeting guide RNAs can be used all at the same time, for example10 or 20 or 30 and so forth, whilst only one (or at least a minimalnumber) of effector protein molecules need to be delivered, as acomparatively small number of effector protein molecules can be usedwith a large number modified guides. The adaptor protein may beassociated (preferably linked or fused to) one or more activators or oneor more repressors. For example, the adaptor protein may be associatedwith a first activator and a second activator. The first and secondactivators may be the same, but they are preferably differentactivators. Three or more or even four or more activators (orrepressors) may be used, but package size may limit the number beinghigher than 5 different functional domains. Linkers are preferably used,over a direct fusion to the adaptor protein, where two or morefunctional domains are associated with the adaptor protein. Suitablelinkers might include the GlySer linker.

It is also envisaged that the nucleic acid-targeting effectorprotein-guide RNA complex as a whole may be associated with two or morefunctional domains. For example, there may be two or more functionaldomains associated with the nucleic acid-targeting effector protein, orthere may be two or more functional domains associated with the guideRNA (via one or more adaptor proteins), or there may be one or morefunctional domains associated with the nucleic acid-targeting effectorprotein and one or more functional domains associated with the guide RNA(via one or more adaptor proteins).

The fusion between the adaptor protein and the activator or repressormay include a linker. For example, GlySer linkers GGGS can be used. Theycan be used in repeats of 3 ((GGGGS)₃) or 6, 9 or even 12 or more, toprovide suitable lengths, as required. Linkers can be used between theguide RNAs and the functional domain (activator or repressor), orbetween the nucleic acid-targeting effector protein and the functionaldomain (activator or repressor). The linkers the user to engineerappropriate amounts of “mechanical flexibility”.

The invention comprehends a nucleic acid-targeting complex comprising anucleic acid-targeting effector protein and a guide RNA, wherein thenucleic acid-targeting effector protein comprises at least one mutation,such that the nucleic acid-targeting Cas protein has no more than 5% ofthe activity of the nucleic acid-targeting Cas protein not having the atleast one mutation and, optionally, at least one or more nuclearlocalization sequences; the guide RNA comprises a guide sequence capableof hybridizing to a target sequence in a RNA of interest in a cell; andwherein: the nucleic acid-targeting effector protein is associated withtwo or more functional domains; or at least one loop of the guide RNA ismodified by the insertion of distinct RNA sequence(s) that bind to oneor more adaptor proteins, and wherein the adaptor protein is associatedwith two or more functional domains; or the nucleic acid-targetingeffector protein is associated with one or more functional domains andat least one loop of the guide RNA is modified by the insertion ofdistinct RNA sequence(s) that bind to one or more adaptor proteins, andwherein the adaptor protein is associated with one or more functionaldomains.

Delivery Generally C2c2 Effector Protein Complexes can DeliverFunctional Effectors

Unlike CRISPR-Cas-mediated gene knockout, which permanently eliminatesexpression by mutating the gene at the DNA level, CRISPR-Cas knockdownallows for temporary reduction of gene expression through the use ofartificial transcription or translation factors. Mutating key residuesin both DNA or RNA cleavage domains of the C2c2 protein results in thegeneration of a catalytically inactive C2c2. A catalytically inactiveC2c2 complexes with a guide RNA and localizes to the DNA or RNA sequencespecified by that guide RNA's targeting domain, however, it does notcleave the target DNA or RNA. Fusion of the inactive C2c2 protein to aneffector domain, e.g., a transcription or translation repression domain,enables recruitment of the effector to any DNA or RNA site specified bythe guide RNA. In certain embodiments, C2c2 may be fused to atranscriptional repression domain and recruited to the promoter regionof a gene. Especially for gene repression, it is contemplated hereinthat blocking the binding site of an endogenous transcription factorwould aid in downregulating gene expression. In another embodiment, aninactive C2c2 can be fused to a chromatin modifying protein. Alteringchromatin status can result in decreased expression of the target gene.In further embodiments, C2c2 may be fused to a translation repressiondomain.

In an embodiment, a guide RNA molecule can be targeted to a knowntranscription response elements (e.g., promoters, enhancers, etc.), aknown upstream activating sequences, and/or sequences of unknown orknown function that are suspected of being able to control expression ofthe target DNA.

In some methods, a target polynucleotide can be inactivated to effectthe modification of the expression in a cell. For example, upon thebinding of a CRISPR complex to a target sequence in a cell, the targetpolynucleotide is inactivated such that the sequence is not transcribed,the coded protein is not produced, or the sequence does not function asthe wild-type sequence does. For example, a protein or microRNA codingsequence may be inactivated such that the protein is not produced. Insome methods, a target polynucleotide can be inactivated to effect themodification of the expression in a cell. For example, upon the bindingof a CRISPR complex to an RNA target sequence in a cell, the targetpolynucleotide is inactivated such that the sequence is not translated,affecting the expression level of the protein in the cell.

In particular embodiments, the CRISPR enzyme comprises one or moremutations selected from the group consisting of R597A, H602A, R1278A andH1283A and/or the one or more mutations are in the HEPN domain of theCRISPR enzyme or is a mutation as otherwise discussed herein. In someembodiments, the CRISPR enzyme has one or more mutations in a catalyticdomain, wherein when transcribed, the direct repeat sequence forms asingle stem loop and the guide sequence directs sequence-specificbinding of a CRISPR complex to the target sequence, and wherein theenzyme further comprises a functional domain. In some embodiments, thefunctional domain is a. In some embodiments, the functional domain is atranscription repression domain, preferably KRAB. In some embodiments,the transcription repression domain is SID, or concatemers of SID (egSID4X). In some embodiments, the functional domain is an epigeneticmodifying domain, such that an epigenetic modifying enzyme is provided.In some embodiments, the functional domain is an activation domain,which may be the P65 activation domain.

Delivery of the C2c2 Effector Protein Complex or Components Thereof

Through this disclosure and the knowledge in the art, TALEs, CRISPR-Cassystems, or components thereof or nucleic acid molecules thereof ornucleic acid molecules encoding or providing components thereof may bedelivered by a delivery system herein described both generally and indetail.

Vector delivery, e.g., plasmid, viral delivery: The CRISPR enzyme, forinstance a Type V protein such as C2c2, and/or any of the present RNAs,for instance a guide RNA, can be delivered using any suitable vector,e.g., plasmid or viral vectors, such as adeno associated virus (AAV),lentivirus, adenovirus or other viral vector types, or combinationsthereof. Effector proteins and one or more guide RNAs can be packagedinto one or more vectors, e.g., plasmid or viral vectors. In someembodiments, the vector, e.g., plasmid or viral vector is delivered tothe tissue of interest by, for example, an intramuscular injection,while other times the delivery is via intravenous, transdermal,intranasal, oral, mucosal, or other delivery methods. Such delivery maybe either via a single dose, or multiple doses. One skilled in the artunderstands that the actual dosage to be delivered herein may varygreatly depending upon a variety of factors, such as the vector choice,the target cell, organism, or tissue, the general condition of thesubject to be treated, the degree of transformation/modification sought,the administration route, the administration mode, the type oftransformation/modification sought, etc.

Such a dosage may further contain, for example, a carrier (water,saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin,dextran, agar, pectin, peanut oil, sesame oil, etc.), a diluent, apharmaceutically-acceptable carrier (e.g., phosphate-buffered saline), apharmaceutically-acceptable excipient, and/or other compounds known inthe art. The dosage may further contain one or more pharmaceuticallyacceptable salts such as, for example, a mineral acid salt such as ahydrochloride, a hydrobromide, a phosphate, a sulfate, etc.; and thesalts of organic acids such as acetates, propionates, malonates,benzoates, etc. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, gels or gelling materials,flavorings, colorants, microspheres, polymers, suspension agents, etc.may also be present herein. In addition, one or more other conventionalpharmaceutical ingredients, such as preservatives, humectants,suspending agents, surfactants, antioxidants, anticaking agents,fillers, chelating agents, coating agents, chemical stabilizers, etc.may also be present, especially if the dosage form is a reconstitutableform. Suitable exemplary ingredients include microcrystalline cellulose,carboxymethylcellulose sodium, polysorbate 80, phenylethyl alcohol,chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propylgallate, the parabens, ethyl vanillin, glycerin, phenol,parachlorophenol, gelatin, albumin and a combination thereof. A thoroughdiscussion of pharmaceutically acceptable excipients is available inREMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991) which isincorporated by reference herein.

In an embodiment herein the delivery is via an adenovirus, which may beat a single booster dose containing at least 1×10⁵ particles (alsoreferred to as particle units, pu) of adenoviral vector. In anembodiment herein, the dose preferably is at least about 1×10⁶ particles(for example, about 1×10⁶-1×10¹² particles), more preferably at leastabout 1×10⁷ particles, more preferably at least about 1×10⁸ particles(e.g., about 1×10⁸-1×10¹¹ particles or about 1×10⁸-1×10¹² particles),and most preferably at least about 1×10⁰ particles (e.g., about1×10⁹-1×10¹⁰ particles or about 1×10⁹-1×10¹² particles), or even atleast about 1×10¹⁰ particles (e.g., about 1×10¹⁰-1×10¹² particles) ofthe adenoviral vector. Alternatively, the dose comprises no more thanabout 1×10¹⁴ particles, preferably no more than about 1×10¹³ particles,even more preferably no more than about 1×10¹² particles, even morepreferably no more than about 1×10¹¹ particles, and most preferably nomore than about 1×10¹⁰ particles (e.g., no more than about 1×10⁹articles). Thus, the dose may contain a single dose of adenoviral vectorwith, for example, about 1×10⁶ particle units (pu), about 2×10⁶ pu,about 4×10⁶ pu, about 1×10⁷ pu, about 2×10⁷ pu, about 4×10⁷ pu, about1×10⁸ pu, about 2×10⁸ pu, about 4×10⁸ pu, about 1×10⁹ pu, about 2×10⁹pu, about 4×10⁹ pu, about 1×10¹⁰ pu, about 2×10¹⁰ pu, about 4×10¹⁰ pu,about 1×10¹¹ pu, about 2×10¹¹ pu, about 4×10¹¹ pu, about 1×10¹² pu,about 2×10¹² pu, or about 4×10¹² pu of adenoviral vector. See, forexample, the adenoviral vectors in U.S. Pat. No. 8,454,972 B2 to Nabel,et. al., granted on Jun. 4, 2013; incorporated by reference herein, andthe dosages at col 29, lines 36-58 thereof. In an embodiment herein, theadenovirus is delivered via multiple doses.

In an embodiment herein, the delivery is via an AAV. A therapeuticallyeffective dosage for in vivo delivery of the AAV to a human is believedto be in the range of from about 20 to about 50 ml of saline solutioncontaining from about 1×10¹⁰ to about 1×10¹⁰ functional AAV/ml solution.The dosage may be adjusted to balance the therapeutic benefit againstany side effects. In an embodiment herein, the AAV dose is generally inthe range of concentrations of from about 1×10⁵ to 1×10⁵ genomes AAV,from about 1×10⁸ to 1×10²⁰ genomes AAV, from about 1×10¹⁰ to about1×10¹⁶ genomes, or about 1×10¹¹ to about 1×10¹⁶ genomes AAV. A humandosage may be about 1×10¹³ genomes AAV. Such concentrations may bedelivered in from about 0.001 ml to about 100 ml, about 0.05 to about 50ml, or about 10 to about 25 ml of a carrier solution. Other effectivedosages can be readily established by one of ordinary skill in the artthrough routine trials establishing dose response curves. See, forexample, U.S. Pat. No. 8,404,658 B2 to Hajjar, et al., granted on Mar.26, 2013, at col. 27, lines 45-60.

In an embodiment herein the delivery is via a plasmid. In such plasmidcompositions, the dosage should be a sufficient amount of plasmid toelicit a response. For instance, suitable quantities of plasmid DNA inplasmid compositions can be from about 0.1 to about 2 mg, or from about1 μg to about 10 μg per 70 kg individual. Plasmids of the invention willgenerally comprise (i) a promoter; (ii) a sequence encoding an nucleicacid-targeting CRISPR enzyme, operably linked to said promoter; (iii) aselectable marker; (iv) an origin of replication; and (v) atranscription terminator downstream of and operably linked to (ii). Theplasmid can also encode the RNA components of a CRISPR complex, but oneor more of these may instead be encoded on a different vector.

The doses herein are based on an average 70 kg individual. The frequencyof administration is within the ambit of the medical or veterinarypractitioner (e.g., physician, veterinarian), or scientist skilled inthe art. It is also noted that mice used in experiments are typicallyabout 20 g and from mice experiments one can scale up to a 70 kgindividual.

In some embodiments the RNA molecules of the invention are delivered inliposome or lipofectin formulations and the like and can be prepared bymethods well known to those skilled in the art. Such methods aredescribed, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and5,580,859, which are herein incorporated by reference. Delivery systemsaimed specifically at the enhanced and improved delivery of siRNA intomammalian cells have been developed, (see, for example, Shen et al FEBSLet. 2003, 539:111-114; Xia et al., Nat. Biotech. 2002, 20:1006-1010;Reich et al., Mol. Vision. 2003, 9: 210-216; Sorensen et al., J. Mol.Biol. 2003, 327: 761-766; Lewis et al., Nat. Gen. 2002, 32: 107-108 andSimeoni et al., NAR 2003, 31, 11: 2717-2724) and may be applied to thepresent invention. siRNA has recently been successfully used forinhibition of gene expression in primates (see for example. Tolentino etal., Retina 24(4):660 which may also be applied to the presentinvention.

Indeed, RNA delivery is a useful method of in vivo delivery. It ispossible to deliver nucleic acid-targeting Cas proteinCas9 and guideRNAgRNA (and, for instance, HR repair template) into cells usingliposomes or particles. Thus delivery of the nucleic acid-targeting Casprotein/CRISPR enzyme, such as a CasCas9 and/or delivery of the guideRNAs of the invention may be in RNA form and via microvesicles,liposomes or particles. For example, Cas mRNA and guide RNA can bepackaged into liposomal particles for delivery in vivo. Liposomaltransfection reagents such as lipofectamine from Life Technologies andother reagents on the market can effectively deliver RNA molecules intothe liver.

Means of delivery of RNA also preferred include delivery of RNA viananoparticles (Cho, S., Goldberg, M., Son, S., Xu, Q., Yang, F., Mei,Y., Bogatyrev, S., Langer, R. and Anderson, D., Lipid-like nanoparticlesfor small interfering RNA delivery to endothelial cells, AdvancedFunctional Materials, 19: 3112-3118, 2010) or exosomes (Schroeder, A.,Levins, C., Cortez, C., Langer, R., and Anderson, D., Lipid-basednanotherapeutics for siRNA delivery, Journal of Internal Medicine, 267:9-21, 2010, PMID: 20059641). Indeed, exosomes have been shown to beparticularly useful in delivery siRNA, a system with some parallels tothe RNA-targeting system. For instance, El-Andaloussi S, et al.(“Exosome-mediated delivery of siRNA in vitro and in vivo.” Nat Protoc.2012 December; 7(12):2112-26. doi: 10.1038/nprot.2012.131. Epub 2012Nov. 15.) describe how exosomes are promising tools for drug deliveryacross different biological barriers and can be harnessed for deliveryof siRNA in vitro and in vivo. Their approach is to generate targetedexosomes through transfection of an expression vector, comprising anexosomal protein fused with a peptide ligand. The exosomes are thenpurify and characterized from transfected cell supernatant, then RNA isloaded into the exosomes. Delivery or administration according to theinvention can be performed with exosomes, in particular but not limitedto the brain. Vitamin E (α-tocopherol) may be conjugated with nucleicacid-targeting Cas protein and delivered to the brain along with highdensity lipoprotein (HDL), for example in a similar manner as was doneby Uno et al. (HUMAN GENE THERAPY 22:711-719 (June 2011)) for deliveringshort-interfering RNA (siRNA) to the brain. Mice were infused viaOsmotic minipumps (model 1007D; Alzet, Cupertino, Calif.) filled withphosphate-buffered saline (PBS) or free TocsiBACE or Toc-siBACE/HDL andconnected with Brain Infusion Kit 3 (Alzet). A brain-infusion cannulawas placed about 0.5 mm posterior to the bregma at midline for infusioninto the dorsal third ventricle. Uno et al. found that as little as 3nmol of Toc-siRNA with HDL could induce a target reduction in comparabledegree by the same ICV infusion method. A similar dosage of nucleicacid-targeting effector protein conjugated to α-tocopherol andco-administered with HDL targeted to the brain may be contemplated forhumans in the present invention, for example, about 3 nmol to about 3μmol of nucleic acid-targeting effector protein targeted to the brainmay be contemplated. Zou et al. ((HUMAN GENE THERAPY 22:465-475 (April2011)) describes a method of lentiviral-mediated delivery ofshort-hairpin RNAs targeting PKCγ for in vivo gene silencing in thespinal cord of rats. Zou et al. administered about 10 μl of arecombinant lentivirus having a titer of 1×10⁹ transducing units (TU)/mlby an intrathecal catheter. A similar dosage of nucleic acid-targetingeffector protein expressed in a lentiviral vector targeted to the brainmay be contemplated for humans in the present invention, for example,about 10-50 ml of nucleic acid-targeting effector protein targeted tothe brain in a lentivirus having a titer of 1×10⁹ transducing units(TU)/ml may be contemplated.

In terms of local delivery to the brain, this can be achieved in variousways. For instance, material can be delivered intrastriatally e.g., byinjection. Injection can be performed stereotactically via a craniotomy.

Enhancing NHEJ or HR efficiency is also helpful for delivery. It ispreferred that NHEJ efficiency is enhanced by co-expressingend-processing enzymes such as Trex2 (Dumitrache et al. Genetics. 2011August; 188(4): 787-797). It is preferred that HR efficiency isincreased by transiently inhibiting NHEJ machineries such as Ku70 andKu86. HR efficiency can also be increased by co-expressing prokaryoticor eukaryotic homologous recombination enzymes such as RecBCD, RecA.

Packing and Promoters Generally

Ways to package nucleic acid-targeting effector protein (such as a TypeV protein such as C2c2) coding nucleic acid molecules, e.g., DNA, intovectors, e.g., viral vectors, to mediate genome modification in vivoinclude:

To achieve NHEJ-mediated gene knockout:

Single virus vector:

Vector containing two or more expression cassettes:

Promoter-nucleic acid-targeting effector protein coding nucleic acidmolecule-terminator

Promoter-guide RNA1-terminator

Promoter-guide RNA (N)-terminator (up to size limit of vector)

Double Virus Vector:

Vector 1 containing one expression cassette for driving the expressionof nucleic acid-targeting effector protein (such as a Type V proteinsuch as C2c2)

Promoter-nucleic acid-targeting effector protein coding nucleic acidmolecule-terminator

Vector 2 containing one more expression cassettes for driving theexpression of one or more guideRNAs

Promoter-guide RNA1-terminator

Promoter-guide RNA1 (N)-terminator (up to size limit of vector)

To mediate homology-directed repair.

-   -   In addition to the single and double virus vector approaches        described above, an additional vector is used to deliver a        homology-direct repair template.

The promoter used to drive nucleic acid-targeting effector protein (suchas a Type V protein such as C2c2) coding nucleic acid moleculeexpression can include:

AAV ITR can serve as a promoter: this is advantageous for eliminatingthe need for an additional promoter element (which can take up space inthe vector). The additional space freed up can be used to drive theexpression of additional elements (gRNA, etc.). Also, ITR activity isrelatively weaker, so can be used to reduce potential toxicity due toover expression of nucleic acid-targeting effector protein (such as aType V protein such as C2c2).

For ubiquitous expression, can use promoters: CMV, CAG, CBh, PGK, SV40,Ferritin heavy or light chains, etc.

For brain or other CNS expression, can use promoters: SynapsinI for allneurons, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or VGAT forGABAergic neurons, etc.

For liver expression, can use Albumin promoter.

For lung expression, can use SP-B.

For endothelial cells, can use ICAM.

For hematopoietic cells can use IFNbeta or CD45.

For Osteoblasts can use OG-2.

The promoter used to drive guide RNA can include:

Pol III promoters such as U6 or H1

Use of Pol II promoter and intronic cassettes to express guide RNA

Adeno Associated Virus (AAV)

nucleic acid-targeting effector protein (such as a Type V protein suchas C2c2) and one or more guide RNA can be delivered using adenoassociated virus (AAV), lentivirus, adenovirus or other plasmid or viralvector types, in particular, using formulations and doses from, forexample, U.S. Pat. No. 8,454,972 (formulations, doses for adenovirus),U.S. Pat. No. 8,404,658 (formulations, doses for AAV) and U.S. Pat. No.5,846,946 (formulations, doses for DNA plasmids) and from clinicaltrials and publications regarding the clinical trials involvinglentivirus, AAV and adenovirus. For examples, for AAV, the route ofadministration, formulation and dose can be as in U.S. Pat. No.8,454,972 and as in clinical trials involving AAV. For Adenovirus, theroute of administration, formulation and dose can be as in U.S. Pat. No.8,404,658 and as in clinical trials involving adenovirus. For plasmiddelivery, the route of administration, formulation and dose can be as inU.S. Pat. No. 5,846,946 and as in clinical studies involving plasmids.Doses may be based on or extrapolated to an average 70 kg individual(e.g., a male adult human), and can be adjusted for patients, subjects,mammals of different weight and species. Frequency of administration iswithin the ambit of the medical or veterinary practitioner (e.g.,physician, veterinarian), depending on usual factors including the age,sex, general health, other conditions of the patient or subject and theparticular condition or symptoms being addressed. The viral vectors canbe injected into the tissue of interest. For cell-type specificgenome/transcriptome modification, the expression of nucleicacid-targeting effector protein (such as a Type V protein such as C2c2)can be driven by a cell-type specific promoter. For example,liver-specific expression might use the Albumin promoter andneuron-specific expression (e.g., for targeting CNS disorders) might usethe Synapsin I promoter.

In terms of in vivo delivery, AAV is advantageous over other viralvectors for a couple of reasons:

-   -   Low toxicity (this may be due to the purification method not        requiring ultra centrifugation of cell particles that can        activate the immune response) and    -   Low probability of causing insertional mutagenesis because it        doesn't integrate into the host genome.

AAV has a packaging limit of 4.5 or 4.75 Kb. This means that nucleicacid-targeting effector protein (such as a Type V protein such as C2c2)as well as a promoter and transcription terminator have to be all fitinto the same viral vector. Therefore embodiments of the inventioninclude utilizing homologs of nucleic acid-targeting effector protein(such as a Type V protein such as C2c2) that are shorter.

As to AAV, the AAV can be AAV1, AAV2, AAV5 or any combination thereof.One can select the AAV of the AAV with regard to the cells to betargeted; e.g., one can select AAV serotypes 1, 2, 5 or a hybrid capsidAAV1, AAV2, AAV5 or any combination thereof for targeting brain orneuronal cells; and one can select AAV4 for targeting cardiac tissue.AAV8 is useful for delivery to the liver. The herein promoters andvectors are preferred individually. A tabulation of certain AAVserotypes as to these cells (see Grimm, D. et al, J. Virol. 82:5887-5911 (2008)) is as follows:

Cell Line AAV-1 AAV-2 AAV-3 AAV-4 AAV-5 AAV-6 AAV-8 AAV-9 Huh-7 13 1002.5 0.0 0.1 10 0.7 0.0 HEK293 25 100 2.5 0.1 0.1 5 0.7 0.1 HeLa 3 1002.0 0.1 6.7 1 0.2 0.1 HepG2 3 100 16.7 0.3 1.7 5 0.3 ND Hep1A 20 100 0.21.0 0.1 1 0.2 0.0 911 17 100 11 0.2 0.1 17 0.1 ND CHO 100 100 14 1.4 33350 10 1.0 COS 33 100 33 3.3 5.0 14 2.0 0.5 MeWo 10 100 20 0.3 6.7 10 1.00.2 NIH3T3 10 100 2.9 2.9 0.3 10 0.3 ND A549 14 100 20 ND 0.5 10 0.5 0.1HT1180 20 100 10 0.1 0.3 33 0.5 0.1 Monocytes 1111 100 ND ND 125 1429 NDND Immature DC 2500 100 ND ND 222 2857 ND ND Mature DC 2222 100 ND ND333 3333 ND ND

Lentivirus

Lentiviruses are complex retroviruses that have the ability to infectand express their genes in both mitotic and post-mitotic cells. The mostcommonly known lentivirus is the human immunodeficiency virus (HIV),which uses the envelope glycoproteins of other viruses to target a broadrange of cell types.

Lentiviruses may be prepared as follows. After cloning pCasES10 (whichcontains a lentiviral transfer plasmid backbone), HEK293FT at lowpassage (p=5) were seeded in a T-75 flask to 50% confluence the daybefore transfection in DMEM with 10% fetal bovine serum and withoutantibiotics. After 20 hours, media was changed to OptiMEM (serum-free)media and transfection was done 4 hours later. Cells were transfectedwith 10 μg of lentiviral transfer plasmid (pCasES10) and the followingpackaging plasmids: 5 μg of pMD2. G (VSV-g pseudotype), and 7.5 ug ofpsPAX2 (gag/pol/rev/tat). Transfection was done in 4 mL OptiMEM with acationic lipid delivery agent (50 uL Lipofectamine 2000 and 100 ul Plusreagent). After 6 hours, the media was changed to antibiotic-free DMEMwith 10% fetal bovine serum. These methods use serum during cellculture, but serum-free methods are preferred.

Lentivirus may be purified as follows. Viral supernatants were harvestedafter 48 hours. Supernatants were first cleared of debris and filteredthrough a 0.45 um low protein binding (PVDF) filter. They were then spunin a ultracentrifuge for 2 hours at 24,000 rpm. Viral pellets wereresuspended in 50 ul of DMEM overnight at 4 C. They were then aliquottedand immediately frozen at −80° C.

In another embodiment, minimal non-primate lentiviral vectors based onthe equine infectious anemia virus (EIAV) are also contemplated,especially for ocular gene therapy (see, e.g., Balagaan, J Gene Med2006; 8: 275-285). In another embodiment, RetinoStat®, an equineinffctious anemia virus-based lentiviral gene therapy vector thatexpresses angiostatic proteins endostatin and angiostatin that isdelivered via a subretinal injection for the treatment of the web formof age-related macular degeneration is also contemplated (see, e.g.,Binley et al., HUMAN GENE THERAPY 23:980-991 (September 2012)) and thisvector may be modified for the nucleic acid-targeting system of thepresent invention.

In another embodiment, self-inactivating lentiviral vectors with ansiRNA targeting a common exon shared by HIV tat/rev, anucleolar-localizing TAR decoy, and an anti-CCR5-specific hammerheadribozyme (see, e.g., DiGiusto et al. (2010) Sci Transl Med 2:36ra43) maybe used/and or adapted to the nucleic acid-targeting system of thepresent invention. A minimum of 2.5×10⁶ CD34+ cells per kilogram patientweight may be collected and prestimulated for 16 to 20 hours in X-VIVO15 medium (Lonza) containing 2 μmol/L-glutamine, stem cell factor (100ng/ml), Flt-3 ligand (Flt-3L) (100 ng/ml), and thrombopoietin (10 ng/ml)(CellGenix) at a density of 2×10⁶ cells/ml. Prestimulated cells may betransduced with lentiviral at a multiplicity of infection of 5 for 16 to24 hours in 75-cm² tissue culture flasks coated with fibronectin (25mg/cm²) (RetroNectin, Takara Bio Inc.).

Lentiviral vectors have been disclosed as in the treatment forParkinson's Disease, see, e.g., US Patent Publication No. 20120295960and U.S. Pat. Nos. 7,303,910 and 7,351,585. Lentiviral vectors have alsobeen disclosed for the treatment of ocular diseases, see e.g., US PatentPublication Nos. 20060281180, 20090007284, US20110117189; US20090017543;US20070054961, US20100317109. Lentiviral vectors have also beendisclosed for delivery to the brain, see, e.g., US Patent PublicationNos. US20110293571; US20110293571, US20040013648, US20070025970,US20090111106 and U.S. Pat. No. 7,259,015.

RNA Delivery

RNA delivery: The nucleic acid-targeting Cas protein, for instance aType V protein such as C2c2, and/or guide RNA, can also be delivered inthe form of RNA. nucleic acid-targeting Cas protein (such as a Type VIprotein such as C2c2) mRNA can be generated using in vitrotranscription. For example, nucleic acid-targeting effector protein(such as a Type V protein such as C2c2) mRNA can be synthesized using aPCR cassette containing the following elements: T7_promoter-kozaksequence (GCCACC)-effector protrein-3′ UTR from beta globin-polyA tail(a string of 120 or more adenines). The cassette can be used fortranscription by T7 polymerase. Guide RNAs can also be transcribed usingin vitro transcription from a cassette containing T7_promoter-GG-guideRNA sequence.

To enhance expression and reduce possible toxicity, the nucleicacid-targeting effector protein-coding sequence and/or the guide RNA canbe modified to include one or more modified nucleoside e.g., usingpseudo-U or 5-Methyl-C.

mRNA delivery methods are especially promising for liver deliverycurrently.

Much clinical work on RNA delivery has focused on RNAi or antisense, butthese systems can be adapted for delivery of RNA for implementing thepresent invention. References below to RNAi etc. should be readaccordingly.

Particle Delivery Systems and/or Formulations:

Several types of particle delivery systems and/or formulations are knownto be useful in a diverse spectrum of biomedical applications. Ingeneral, a particle is defined as a small object that behaves as a wholeunit with respect to its transport and properties. Particles are furtherclassified according to diameter. Coarse particles cover a range between2,500 and 10,000 nanometers. Fine particles are sized between 100 and2,500 nanometers. Ultrafine particles, or nanoparticles, are generallybetween 1 and 100 nanometers in size. The basis of the 100-nm limit isthe fact that novel properties that differentiate particles from thebulk material typically develop at a critical length scale of under 100nm.

As used herein, a particle delivery system/formulation is defined as anybiological delivery system/formulation which includes a particle inaccordance with the present invention. A particle in accordance with thepresent invention is any entity having a greatest dimension (e.g.diameter) of less than 100 microns (μm). In some embodiments, inventiveparticles have a greatest dimension of less than 10 μm. In someembodiments, inventive particles have a greatest dimension of less than2000 nanometers (nm). In some embodiments, inventive particles have agreatest dimension of less than 1000 nanometers (nm). In someembodiments, inventive particles have a greatest dimension of less than900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100nm. Typically, inventive particles have a greatest dimension (e.g.,diameter) of 500 nm or less. In some embodiments, inventive particleshave a greatest dimension (e.g., diameter) of 250 nm or less. In someembodiments, inventive particles have a greatest dimension (e.g.,diameter) of 200 nm or less. In some embodiments, inventive particleshave a greatest dimension (e.g., diameter) of 150 nm or less. In someembodiments, inventive particles have a greatest dimension (e.g.,diameter) of 100 nm or less. Smaller particles, e.g., having a greatestdimension of 50 nm or less are used in some embodiments of theinvention. In some embodiments, inventive particles have a greatestdimension ranging between 25 nm and 200 nm.

Particle characterization (including e.g., characterizing morphology,dimension, etc.) is done using a variety of different techniques. Commontechniques are electron microscopy (TEM, SEM), atomic force microscopy(AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy(XPS), powder X-ray diffraction (XRD), Fourier transform infraredspectroscopy (FTIR), matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MALDI-TOF), ultraviolet-visiblespectroscopy, dual polarisation interferometry and nuclear magneticresonance (NMR). Characterization (dimension measurements) may be madeas to native particles (i.e., preloading) or after loading of the cargo(herein cargo refers to e.g., one or more components of CRISPR-Cassystem e.g., CRISPR enzyme or mRNA or guide RNA, or any combinationthereof, and may include additional carriers and/or excipients) toprovide particles of an optimal size for delivery for any in vitro, exvivo and/or in vivo application of the present invention. In certainpreferred embodiments, particle dimension (e.g., diameter)characterization is based on measurements using dynamic laser scattering(DLS). Mention is made of U.S. Pat. No. 8,709,843; U.S. Pat. No.6,007,845; U.S. Pat. No. 5,855,913; U.S. Pat. No. 5,985,309; U.S. Pat.No. 5,543,158; and the publication by James E. Dahlman and Carmen Barneset al. Nature Nanotechnology (2014) published online 11 May 2014,doi:10.1038/nnano.2014.84, concerning particles, methods of making andusing them and measurements thereof.

Particles delivery systems within the scope of the present invention maybe provided in any form, including but not limited to solid, semi-solid,emulsion, or colloidal particles. As such any of the delivery systemsdescribed herein, including but not limited to, e.g., lipid-basedsystems, liposomes, micelles, microvesicles, exosomes, or gene gun maybe provided as particle delivery systems within the scope of the presentinvention.

Particles

CRISPR enzyme mRNA and guide RNA may be delivered simultaneously usingparticles or lipid envelopes; for instance, CRISPR enzyme and RNA of theinvention, e.g., as a complex, can be delivered via a particle as inDahlman et al., WO2015089419 A2 and documents cited therein, such as 7C1(see, e.g., James E. Dahlman and Carmen Barnes et al. NatureNanotechnology (2014) published online 11 May 2014,doi:10.1038/nnano.2014.84), e.g., delivery particle comprising lipid orlipidoid and hydrophilic polymer, e.g., cationic lipid and hydrophilicpolymer, for instance wherein the cationic lipid comprises1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC) and/or whereinthe hydrophilic polymer comprises ethylene glycol or polyethylene glycol(PEG); and/or wherein the particle further comprises cholesterol (e.g.,particle from formulation 1=DOTAP 100, DMPC 0, PEG 0, Cholesterol 0;formulation number 2=DOTAP 90, DMPC 0, PEG 10, Cholesterol 0;formulation number 3=DOTAP 90, DMPC 0, PEG 5, Cholesterol 5), whereinparticles are formed using an efficient, multistep process whereinfirst, effector protein and RNA are mixed together, e.g., at a 1:1 molarratio, e.g., at room temperature, e.g., for 30 minutes, e.g., insterile, nuclease free 1×PBS; and separately, DOTAP, DMPC, PEG, andcholesterol as applicable for the formulation are dissolved in alcohol,e.g., 100% ethanol; and, the two solutions are mixed together to formparticles containing the complexes).

Nucleic acid-targeting effector proteins (such as a Type VI protein suchas C2c2) mRNA and guide RNA may be delivered simultaneously usingparticles or lipid envelopes.

For example, Su X, Fricke J, Kavanagh D G, Irvine D J (“In vitro and invivo mRNA delivery using lipid-enveloped pH-responsive polymernanoparticles” Mol Pharm. 2011 Jun. 6; 8(3):774-87. doi:10.1021/mp100390w. Epub 2011 Apr. 1) describes biodegradable core-shellstructured particles with a poly(β-amino ester) (PBAE) core enveloped bya phospholipid bilayer shell. These were developed for in vivo mRNAdelivery. The pH-responsive PBAE component was chosen to promoteendosome disruption, while the lipid surface layer was selected tominimize toxicity of the polycation core. Such are, therefore, preferredfor delivering RNA of the present invention.

In one embodiment, particles based on self-assembling bioadhesivepolymers are contemplated, which may be applied to oral delivery ofpeptides, intravenous delivery of peptides and nasal delivery ofpeptides, all to the brain. Other embodiments, such as oral absorptionand ocular delivery of hydrophobic drugs are also contemplated. Themolecular envelope technology involves an engineered polymer envelopewhich is protected and delivered to the site of the disease (see, e.g.,Mazza, M. et al. ACSNano, 2013. 7(2): 1016-1026; Siew, A., et al. MolPharm, 2012. 9(1):14-28; Lalatsa, A., et al. J Contr Rel, 2012.161(2):523-36; Lalatsa, A., et al., Mol Pharm, 2012. 9(6):1665-80;Lalatsa, A., et al. Mol Pharm, 2012. 9(6):1764-74; Garrett, N. L., etal. J Biophotonics, 2012. 5(5-6):458-68; Garrett, N. L., et al. J RamanSpect, 2012. 43(5):681-688; Ahmad, S., et al. J Royal Soc Interface2010. 7:S423-33; Uchegbu, I. F. Expert Opin Drug Deliv, 2006.3(5):629-40; Qu, X., et al. Biomacromolecules, 2006. 7(12):3452-9 andUchegbu, I. F., et al. Int J Pharm, 2001. 224:185-199). Doses of about 5mg/kg are contemplated, with single or multiple doses, depending on thetarget tissue.

In one embodiment, particles that can deliver RNA to a cancer cell tostop tumor growth developed by Dan Anderson's lab at MIT may be used/andor adapted to the nucleic acid-targeting system of the presentinvention. In particular, the Anderson lab developed fully automated,combinatorial systems for the synthesis, purification, characterization,and formulation of new biomaterials and nanoformulations. See, e.g.,Alabi et al., Proc Natl Acad Sci USA. 2013 Aug. 6; 110(32):12881-6;Zhang et al., Adv Mater. 2013 Sep. 6; 25(33):4641-5; Jiang et al., NanoLett. 2013 Mar. 13; 13(3):1059-64; Karagiannis et al., ACS Nano. 2012Oct. 23; 6(10):8484-7; Whitehead et al., ACS Nano. 2012 Aug. 28;6(8):6922-9 and Lee et al., Nat Nanotechnol. 2012 Jun. 3; 7(6):389-93.

US patent application 20110293703 relates to lipidoid compounds are alsoparticularly useful in the administration of polynucleotides, which maybe applied to deliver the nucleic acid-targeting system of the presentinvention. In one aspect, the aminoalcohol lipidoid compounds arecombined with an agent to be delivered to a cell or a subject to formmicroparticles, nanoparticles, liposomes, or micelles. The agent to bedelivered by the particles, liposomes, or micelles may be in the form ofa gas, liquid, or solid, and the agent may be a polynucleotide, protein,peptide, or small molecule. The minoalcohol lipidoid compounds may becombined with other aminoalcohol lipidoid compounds, polymers (syntheticor natural), surfactants, cholesterol, carbohydrates, proteins, lipids,etc. to form the particles. These particles may then optionally becombined with a pharmaceutical excipient to form a pharmaceuticalcomposition.

US Patent Publication No. 20110293703 also provides methods of preparingthe aminoalcohol lipidoid compounds. One or more equivalents of an amineare allowed to react with one or more equivalents of anepoxide-terminated compound under suitable conditions to form anaminoalcohol lipidoid compound of the present invention. In certainembodiments, all the amino groups of the amine are fully reacted withthe epoxide-terminated compound to form tertiary amines. In otherembodiments, all the amino groups of the amine are not fully reactedwith the epoxide-terminated compound to form tertiary amines therebyresulting in primary or secondary amines in the aminoalcohol lipidoidcompound. These primary or secondary amines are left as is or may bereacted with another electrophile such as a different epoxide-terminatedcompound. As will be appreciated by one skilled in the art, reacting anamine with less than excess of epoxide-terminated compound will resultin a plurality of different aminoalcohol lipidoid compounds with variousnumbers of tails. Certain amines may be fully functionalized with twoepoxide-derived compound tails while other molecules will not becompletely functionalized with epoxide-derived compound tails. Forexample, a diamine or polyamine may include one, two, three, or fourepoxide-derived compound tails off the various amino moieties of themolecule resulting in primary, secondary, and tertiary amines. Incertain embodiments, all the amino groups are not fully functionalized.In certain embodiments, two of the same types of epoxide-terminatedcompounds are used. In other embodiments, two or more differentepoxide-terminated compounds are used. The synthesis of the aminoalcohollipidoid compounds is performed with or without solvent, and thesynthesis may be performed at higher temperatures ranging from 30-100°C., preferably at approximately 50-90° C. The prepared aminoalcohollipidoid compounds may be optionally purified. For example, the mixtureof aminoalcohol lipidoid compounds may be purified to yield anaminoalcohol lipidoid compound with a particular number ofepoxide-derived compound tails. Or the mixture may be purified to yielda particular stereo- or regioisomer. The aminoalcohol lipidoid compoundsmay also be alkylated using an alkyl halide (e.g., methyl iodide) orother alkylating agent, and/or they may be acylated.

US Patent Publication No. 20110293703 also provides libraries ofaminoalcohol lipidoid compounds prepared by the inventive methods. Theseaminoalcohol lipidoid compounds may be prepared and/or screened usinghigh-throughput techniques involving liquid handlers, robots, microtiterplates, computers, etc. In certain embodiments, the aminoalcohollipidoid compounds are screened for their ability to transfectpolynucleotides or other agents (e.g., proteins, peptides, smallmolecules) into the cell.

US Patent Publication No. 20130302401 relates to a class ofpoly(beta-amino alcohols) (PBAAs) has been prepared using combinatorialpolymerization. The inventive PBAAs may be used in biotechnology andbiomedical applications as coatings (such as coatings of films ormultilayer films for medical devices or implants), additives, materials,excipients, non-biofouling agents, micropatterning agents, and cellularencapsulation agents. When used as surface coatings, these PBAAselicited different levels of inflammation, both in vitro and in vivo,depending on their chemical structures. The large chemical diversity ofthis class of materials allowed us to identify polymer coatings thatinhibit macrophage activation in vitro. Furthermore, these coatingsreduce the recruitment of inflammatory cells, and reduce fibrosis,following the subcutaneous implantation of carboxylated polystyrenemicroparticles. These polymers may be used to form polyelectrolytecomplex capsules for cell encapsulation. The invention may also havemany other biological applications such as antimicrobial coatings, DNAor siRNA delivery, and stem cell tissue engineering. The teachings of USPatent Publication No. 20130302401 may be applied to the nucleicacid-targeting system of the present invention.

In another embodiment, lipid nanoparticles (LNPs) are contemplated. Anantitransthyretin small interfering RNA has been encapsulated in lipidnanoparticles and delivered to humans (see, e.g., Coelho et al., N EnglJ Med 2013; 369:819-29), and such a system may be adapted and applied tothe nucleic acid-targeting system of the present invention. Doses ofabout 0.01 to about 1 mg per kg of body weight administeredintravenously are contemplated. Medications to reduce the risk ofinfusion-related reactions are contemplated, such as dexamethasone,acetampinophen, diphenhydramine or cetirizine, and ranitidine arecontemplated. Multiple doses of about 0.3 mg per kilogram every 4 weeksfor five doses are also contemplated.

LNPs have been shown to be highly effective in delivering siRNAs to theliver (see, e.g., Tabernero et al., Cancer Discovery, April 2013, Vol.3, No. 4, pages 363-470) and are therefore contemplated for deliveringRNA encoding nucleic acid-targeting effector protein to the liver. Adosage of about four doses of 6 mg/kg of the LNP every two weeks may becontemplated. Tabernero et al. demonstrated that tumor regression wasobserved after the first 2 cycles of LNPs dosed at 0.7 mg/kg, and by theend of 6 cycles the patient had achieved a partial response withcomplete regression of the lymph node metastasis and substantialshrinkage of the liver tumors. A complete response was obtained after 40doses in this patient, who has remained in remission and completedtreatment after receiving doses over 26 months. Two patients with RCCand extrahepatic sites of disease including kidney, lung, and lymphnodes that were progressing following prior therapy with VEGF pathwayinhibitors had stable disease at all sites for approximately 8 to 12months, and a patient with PNET and liver metastases continued on theextension study for 18 months (36 doses) with stable disease.

However, the charge of the LNP must be taken into consideration. Ascationic lipids combined with negatively charged lipids to inducenonbilayer structures that facilitate intracellular delivery. Becausecharged LNPs are rapidly cleared from circulation following intravenousinjection, ionizable cationic lipids with pKa values below 7 weredeveloped (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no. 12,pages 1286-2200, December 2011). Negatively charged polymers such as RNAmay be loaded into LNPs at low pH values (e.g., pH 4) where theionizable lipids display a positive charge. However, at physiological pHvalues, the LNPs exhibit a low surface charge compatible with longercirculation times. Four species of ionizable cationic lipids have beenfocused upon, namely 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA), and1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA).It has been shown that LNP siRNA systems containing these lipids exhibitremarkably different gene silencing properties in hepatocytes in vivo,with potencies varying according to the seriesDLinKC2-DMA>DLinKDMA>DLinDMA>>DLinDAP employing a Factor VII genesilencing model (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no.12, pages 1286-2200, December 2011). A dosage of 1 μg/ml of LNP orCRISPR-Cas RNA in or associated with the LNP may be contemplated,especially for a formulation containing DLinKC2-DMA.

Preparation of LNPs and CRISPR-Cas encapsulation may be used/and oradapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages1286-2200, December 2011). The cationic lipids1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA),1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA),(3-o-[2″-(methoxypolyethyleneglycol 2000)succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), andR-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-C-DOMG) may be providedby Tekmira Pharmaceuticals (Vancouver, Canada) or synthesized.Cholesterol may be purchased from Sigma (St Louis, Mo.). The specificnucleic acid-targeting complex (CRISPR-Cas) RNA may be encapsulated inLNPs containing DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationiclipid:DSPC:CHOL:PEGS-DMG or PEG-C-DOMG at 40:10:40:10 molar ratios).When required, 0.2% SP-DiOC18 (Invitrogen, Burlington, Canada) may beincorporated to assess cellular uptake, intracellular delivery, andbiodistribution. Encapsulation may be performed by dissolving lipidmixtures comprised of cationic lipid:DSPC:cholesterol:PEG-c-DOMG(40:10:40:10 molar ratio) in ethanol to a final lipid concentration of10 mmol/l. This ethanol solution of lipid may be added drop-wise to 50mmol/l citrate, pH 4.0 to form multilamellar vesicles to produce a finalconcentration of 30% ethanol vol/vol. Large unilamellar vesicles may beformed following extrusion of multilamellar vesicles through two stacked80 nm Nuclepore polycarbonate filters using the Extruder (NorthernLipids, Vancouver, Canada). Encapsulation may be achieved by adding RNAdissolved at 2 mg/ml in 50 mmol/l citrate, pH 4.0 containing 30% ethanolvol/vol drop-wise to extruded preformed large unilamellar vesicles andincubation at 31° C. for 30 minutes with constant mixing to a finalRNA/lipid weight ratio of 0.06/1 wt/wt. Removal of ethanol andneutralization of formulation buffer were performed by dialysis againstphosphate-buffered saline (PBS), pH 7.4 for 16 hours using Spectra/Por 2regenerated cellulose dialysis membranes. Particle size distribution maybe determined by dynamic light scattering using a NICOMP 370 particlesizer, the vesicle/intensity modes, and Gaussian fitting (NicompParticle Sizing, Santa Barbara, Calif.). The particle size for all threeLNP systems may be ˜70 nm in diameter. RNA encapsulation efficiency maybe determined by removal of free RNA using VivaPureD MiniH columns(Sartorius Stedim Biotech) from samples collected before and afterdialysis. The encapsulated RNA may be extracted from the elutedparticles and quantified at 260 nm. RNA to lipid ratio was determined bymeasurement of cholesterol content in vesicles using the Cholesterol Eenzymatic assay from Wako Chemicals USA (Richmond, Va.). In conjunctionwith the herein discussion of LNPs and PEG lipids, PEGylated liposomesor LNPs are likewise suitable for delivery of a nucleic acid-targetingsystem or components thereof.

Preparation of large LNPs may be used/and or adapted from Rosin et al,Molecular Therapy, vol. 19, no. 12, pages 1286-2200, December 2011. Alipid premix solution (20.4 mg/ml total lipid concentration) may beprepared in ethanol containing DLinKC2-DMA, DSPC, and cholesterol at50:10:38.5 molar ratios. Sodium acetate may be added to the lipid premixat a molar ratio of 0.75:1 (sodium acetate:DLinKC2-DMA). The lipids maybe subsequently hydrated by combining the mixture with 1.85 volumes ofcitrate buffer (10 mmol/l, pH 3.0) with vigorous stirring, resulting inspontaneous liposome formation in aqueous buffer containing 35% ethanol.The liposome solution may be incubated at 37° C. to allow fortime-dependent increase in particle size. Aliquots may be removed atvarious times during incubation to investigate changes in liposome sizeby dynamic light scattering (Zetasizer Nano Z S, Malvern Instruments,Worcestershire, UK). Once the desired particle size is achieved, anaqueous PEG lipid solution (stock=10 mg/ml PEG-DMG in 35% (vol/vol)ethanol) may be added to the liposome mixture to yield a final PEG molarconcentration of 3.5% of total lipid. Upon addition of PEG-lipids, theliposomes should their size, effectively quenching further growth. RNAmay then be added to the empty liposomes at a RNA to total lipid ratioof approximately 1:10 (wt:wt), followed by incubation for 30 minutes at37° C. to form loaded LNPs. The mixture may be subsequently dialyzedovernight in PBS and filtered with a 0.45-μm syringe filter.

Spherical Nucleic Acid (SNA™) constructs and other particles(particularly gold particles) are also contemplated as a means todelivery nucleic acid-targeting system to intended targets. Significantdata show that AuraSense Therapeutics' Spherical Nucleic Acid (SNA™)constructs, based upon nucleic acid-functionalized gold particles, areuseful.

Literature that may be employed in conjunction with herein teachingsinclude: Cutler et al., J. Am. Chem. Soc. 2011 133:9254-9257, Hao etal., Small. 2011 7:3158-3162, Zhang et al., ACS Nano. 2011 5:6962-6970,Cutler et al., J. Am. Chem. Soc. 2012 134:1376-1391, Young et al., NanoLett. 2012 12:3867-71, Zheng et al., Proc. Natl. Acad. Sci. USA. 2012109:11975-80, Mirkin, Nanomedicine 2012 7:635-638 Zhang et al., J. Am.Chem. Soc. 2012 134:16488-1691, Weintraub, Nature 2013 495:S14-S16, Choiet al., Proc. Natl. Acad. Sci. USA. 2013 110(19):7625-7630, Jensen etal., Sci. Transl. Med. 5, 209ra152 (2013) and Mirkin, et al., Small,10:186-192.

Self-assembling particles with RNA may be constructed withpolyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD)peptide ligand attached at the distal end of the polyethylene glycol(PEG). This system has been used, for example, as a means to targettumor neovasculature expressing integrins and deliver siRNA inhibitingvascular endothelial growth factor receptor-2 (VEGF R2) expression andthereby achieve tumor angiogenesis (see, e.g., Schiffelers et al.,Nucleic Acids Research, 2004, Vol. 32, No. 19). Nanoplexes may beprepared by mixing equal volumes of aqueous solutions of cationicpolymer and nucleic acid to give a net molar excess of ionizablenitrogen (polymer) to phosphate (nucleic acid) over the range of 2 to 6.The electrostatic interactions between cationic polymers and nucleicacid resulted in the formation of polyplexes with average particle sizedistribution of about 100 nm, hence referred to here as nanoplexes. Adosage of about 100 to 200 mg of nucleic acid-targeting complex RNA isenvisioned for delivery in the self-assembling particles of Schiffelerset al.

The nanoplexes of Bartlett et al. (PNAS, Sep. 25, 2007, vol. 104, no.39) may also be applied to the present invention. The nanoplexes ofBartlett et al. are prepared by mixing equal volumes of aqueoussolutions of cationic polymer and nucleic acid to give a net molarexcess of ionizable nitrogen (polymer) to phosphate (nucleic acid) overthe range of 2 to 6. The electrostatic interactions between cationicpolymers and nucleic acid resulted in the formation of polyplexes withaverage particle size distribution of about 100 nm, hence referred tohere as nanoplexes. The DOTA-siRNA of Bartlett et al. was synthesized asfollows: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acidmono(N-hydroxysuccinimide ester) (DOTA-NHSester) was ordered fromMacrocyclics (Dallas, Tex.). The amine modified RNA sense strand with a100-fold molar excess of DOTA-NHS-ester in carbonate buffer (pH 9) wasadded to a microcentrifuge tube. The contents were reacted by stirringfor 4 h at room temperature. The DOTA-RNAsense conjugate wasethanol-precipitated, resuspended in water, and annealed to theunmodified antisense strand to yield DOTA-siRNA. All liquids werepretreated with Chelex-100 (Bio-Rad, Hercules, Calif.) to remove tracemetal contaminants. Tf-targeted and nontargeted siRNA particles may beformed by using cyclodextrin-containing polycations. Typically,particles were formed in water at a charge ratio of 3 (+/−) and an siRNAconcentration of 0.5 g/liter. One percent of the adamantane-PEGmolecules on the surface of the targeted particles were modified with Tf(adamantane-PEG-Tf). The particles were suspended in a 5% (wt/vol)glucose carrier solution for injection.

Davis et al. (Nature, Vol 464, 15 Apr. 2010) conducts a RNA clinicaltrial that uses a targeted particle-delivery system (clinical trialregistration number NCT00689065). Patients with solid cancers refractoryto standard-of-care therapies are administered doses of targetedparticles on days 1, 3, 8 and 10 of a 21-day cycle by a 30-minintravenous infusion. The particles comprise, consist essentially of, orconsist of a synthetic delivery system containing: (1) a linear,cyclodextrin-based polymer (CDP), (2) a human transferrin protein (TF)targeting ligand displayed on the exterior of the nanoparticle to engageTF receptors (TFR) on the surface of the cancer cells, (3) a hydrophilicpolymer (polyethylene glycol (PEG) used to promote nanoparticlestability in biological fluids), and (4) siRNA designed to reduce theexpression of the RRM2 (sequence used in the clinic was previouslydenoted siR2B+5). The TFR has long been known to be upregulated inmalignant cells, and RRM2 is an established anti-cancer target. Theseparticles (clinical version denoted as CALAA-01) have been shown to bewell tolerated in multi-dosing studies in non-human primates. Although asingle patient with chronic myeloid leukaemia has been administeredsiRNA by liposomal delivery, Davis et al.'s clinical trial is theinitial human trial to systemically deliver siRNA with a targeteddelivery system and to treat patients with solid cancer. To ascertainwhether the targeted delivery system can provide effective delivery offunctional siRNA to human tumours, Davis et al. investigated biopsiesfrom three patients from three different dosing cohorts; patients A, Band C, all of whom had metastatic melanoma and received CALAA-01 dosesof 18, 24 and 30 mg m⁻² siRNA, respectively. Similar doses may also becontemplated for the nucleic acid-targeting system of the presentinvention. The delivery of the invention may be achieved with particlescontaining a linear, cyclodextrin-based polymer (CDP), a humantransferrin protein (TF) targeting ligand displayed on the exterior ofthe particle to engage TF receptors (TFR) on the surface of the cancercells and/or a hydrophilic polymer (for example, polyethylene glycol(PEG) used to promote particle stability in biological fluids).

In terms of this invention, it is preferred to have one or morecomponents of nucleic acid-targeting complex, e.g., nucleicacid-targeting effector protein or mRNA, or guide RNA delivered usingparticles or lipid envelopes. Other delivery systems or vectors are maybe used in conjunction with the particle aspects of the invention.

In general, a “nanoparticle” refers to any particle having a diameter ofless than 1000 nm. In certain preferred embodiments, nanoparticles ofthe invention have a greatest dimension (e.g., diameter) of 500 nm orless. In other preferred embodiments, nanoparticles of the inventionhave a greatest dimension ranging between 25 nm and 200 nm. In otherpreferred embodiments, particles of the invention have a greatestdimension of 100 nm or less. In other preferred embodiments,nanoparticles of the invention have a greatest dimension ranging between35 nm and 60 nm.

Particles encompassed in the present invention may be provided indifferent forms, e.g., as solid particles (e.g., metal such as silver,gold, iron, titanium), non-metal, lipid-based solids, polymers),suspensions of particles, or combinations thereof. Metal, dielectric,and semiconductor particles may be prepared, as well as hybridstructures (e.g, core-shell particles). Particles made of semiconductingmaterial may also be labeled quantum dots if they are small enough(typically sub 10 nm) that quantization of electronic energy levelsoccurs. Such nanoscale particles are used in biomedical applications asdrug carriers or imaging agents and may be adapted for similar purposesin the present invention.

Semi-solid and soft particles have been manufactured, and are within thescope of the present invention. A prototype particle of semi-solidnature is the liposome. Various types of liposome particles arecurrently used clinically as delivery systems for anticancer drugs andvaccines. Particles with one half hydrophilic and the other halfhydrophobic are termed Janus particles and are particularly effectivefor stabilizing emulsions. They can self-assemble at water/oilinterfaces and act as solid surfactants.

U.S. Pat. No. 8,709,843, incorporated herein by reference, provides adrug delivery system for targeted delivery of therapeuticagent-containing particles to tissues, cells, and intracellularcompartments. The invention provides targeted particles comprisingpolymer conjugated to a surfactant, hydrophilic polymer or lipid.

U.S. Pat. No. 6,007,845, incorporated herein by reference, providesparticles which have a core of a multiblock copolymer formed bycovalently linking a multifunctional compound with one or morehydrophobic polymers and one or more hydrophilic polymers, and contain abiologically active material.

U.S. Pat. No. 5,855,913, incorporated herein by reference, provides aparticulate composition having aerodynamically light particles having atap density of less than 0.4 g/cm3 with a mean diameter of between 5 μmand 30 μm, incorporating a surfactant on the surface thereof for drugdelivery to the pulmonary system.

U.S. Pat. No. 5,985,309, incorporated herein by reference, providesparticles incorporating a surfactant and/or a hydrophilic or hydrophobiccomplex of a positively or negatively charged therapeutic or diagnosticagent and a charged molecule of opposite charge for delivery to thepulmonary system.

U.S. Pat. No. 5,543,158, incorporated herein by reference, providesbiodegradable injectable particles having a biodegradable solid corecontaining a biologically active material and poly(alkylene glycol)moieties on the surface.

WO2012135025 (also published as US20120251560), incorporated herein byreference, describes conjugated polyethyleneimine (PEI) polymers andconjugated aza-macrocycles (collectively referred to as “conjugatedlipomer” or “lipomers”). In certain embodiments, it can be envisionedthat such methods and materials of herein-cited documents, e.g.,conjugated lipomers can be used in the context of the nucleicacid-targeting system to achieve in vitro, ex vivo and in vivo genomicperturbations to modify gene expression, including modulation of proteinexpression.

In one embodiment, the particle may be epoxide-modified lipid-polymer,advantageously 7C1 (see, e.g., James E. Dahlman and Carmen Barnes et al.Nature Nanotechnology (2014) published online 11 May 2014,doi:10.1038/nnano.2014.84). C71 was synthesized by reacting C15epoxide-terminated lipids with PEI600 at a 14:1 molar ratio, and wasformulated with C14PEG2000 to produce particles (diameter between 35 and60 nm) that were stable in PBS solution for at least 40 days.

An epoxide-modified lipid-polymer may be utilized to deliver the nucleicacid-targeting system of the present invention to pulmonary,cardiovascular or renal cells, however, one of skill in the art mayadapt the system to deliver to other target organs. Dosage ranging fromabout 0.05 to about 0.6 mg/kg are envisioned. Dosages over several daysor weeks are also envisioned, with a total dosage of about 2 mg/kg.

Exosomes

Exosomes are endogenous nano-vesicles that transport RNAs and proteins,and which can deliver RNA to the brain and other target organs. Toreduce immunogenicity, Alvarez-Erviti et al. (2011, Nat Biotechnol 29:341) used self-derived dendritic cells for exosome production. Targetingto the brain was achieved by engineering the dendritic cells to expressLamp2b, an exosomal membrane protein, fused to the neuron-specific RVGpeptide. Purified exosomes were loaded with exogenous RNA byelectroporation. Intravenously injected RVG-targeted exosomes deliveredGAPDH siRNA specifically to neurons, microglia, oligodendrocytes in thebrain, resulting in a specific gene knockdown. Pre-exposure to RVGexosomes did not attenuate knockdown, and non-specific uptake in othertissues was not observed. The therapeutic potential of exosome-mediatedsiRNA delivery was demonstrated by the strong mRNA (60%) and protein(62%) knockdown of BACE1, a therapeutic target in Alzheimer's disease.

To obtain a pool of immunologically inert exosomes, Alvarez-Erviti etal. harvested bone marrow from inbred C57BL/6 mice with a homogenousmajor histocompatibility complex (MHC) haplotype. As immature dendriticcells produce large quantities of exosomes devoid of T-cell activatorssuch as MHC-II and CD86, Alvarez-Erviti et al. selected for dendriticcells with granulocyte/macrophage-colony stimulating factor (GM-CSF) for7 d. Exosomes were purified from the culture supernatant the followingday using well-established ultracentrifugation protocols. The exosomesproduced were physically homogenous, with a size distribution peaking at80 nm in diameter as determined by particle tracking analysis (NTA) andelectron microscopy. Alvarez-Erviti et al. obtained 6-12 μg of exosomes(measured based on protein concentration) per 10⁶ cells.

Next, Alvarez-Erviti et al. investigated the possibility of loadingmodified exosomes with exogenous cargoes using electroporation protocolsadapted for nanoscale applications. As electroporation for membraneparticles at the nanometer scale is not well-characterized, nonspecificCy5-labeled RNA was used for the empirical optimization of theelectroporation protocol. The amount of encapsulated RNA was assayedafter ultracentrifugation and lysis of exosomes. Electroporation at 400V and 125 F resulted in the greatest retention of RNA and was used forall subsequent experiments.

Alvarez-Erviti et al. administered 150 μg of each BACE1 siRNAencapsulated in 150 μg of RVG exosomes to normal C57BL/6 mice andcompared the knockdown efficiency to four controls: untreated mice, miceinjected with RVG exosomes only, mice injected with BACE1 siRNAcomplexed to an in vivo cationic liposome reagent and mice injected withBACE1 siRNA complexed to RVG-9R, the RVG peptide conjugated to 9D-arginines that electrostatically binds to the siRNA. Cortical tissuesamples were analyzed 3 d after administration and a significant proteinknockdown (45%, P<0.05, versus 62%, P<0.01) in both siRNA-RVG-9R-treatedand siRNARVG exosome-treated mice was observed, resulting from asignificant decrease in BACE1 mRNA levels (66% [+ or −] 15%, P<0.001 and61% [+ or −] 13% respectively, P<0.01). Moreover, Applicantsdemonstrated a significant decrease (55%, P<0.05) in the total[beta]-amyloid 1-42 levels, a main component of the amyloid plaques inAlzheimer's pathology, in the RVG-exosome-treated animals. The decreaseobserved was greater than the β-amyloid 1-40 decrease demonstrated innormal mice after intraventricular injection of BACE1 inhibitors.Alvarez-Erviti et al. carried out 5′-rapid amplification of cDNA ends(RACE) on BACE1 cleavage product, which provided evidence ofRNAi-mediated knockdown by the siRNA.

Finally, Alvarez-Erviti et al. investigated whether RNA-RVG exosomesinduced immune responses in vivo by assessing IL-6, IP-10, TNFα andIFN-α serum concentrations. Following exosome treatment, nonsignificantchanges in all cytokines were registered similar to siRNA-transfectionreagent treatment in contrast to siRNA-RVG-9R, which potently stimulatedIL-6 secretion, confirming the immunologically inert profile of theexosome treatment. Given that exosomes encapsulate only 20% of siRNA,delivery with RVG-exosome appears to be more efficient than RVG-9Rdelivery as comparable mRNA knockdown and greater protein knockdown wasachieved with fivefold less siRNA without the corresponding level ofimmune stimulation. This experiment demonstrated the therapeuticpotential of RVG-exosome technology, which is potentially suited forlong-term silencing of genes related to neurodegenerative diseases. Theexosome delivery system of Alvarez-Erviti et al. may be applied todeliver the nucleic acid-targeting system of the present invention totherapeutic targets, especially neurodegenerative diseases. A dosage ofabout 100 to 1000 mg of nucleic acid-targeting system encapsulated inabout 100 to 1000 mg of RVG exosomes may be contemplated for the presentinvention.

El-Andaloussi et al. (Nature Protocols 7, 2112-2126(2012)) discloses howexosomes derived from cultured cells can be harnessed for delivery ofRNA in vitro and in vivo. This protocol first describes the generationof targeted exosomes through transfection of an expression vector,comprising an exosomal protein fused with a peptide ligand. Next,El-Andaloussi et al. explain how to purify and characterize exosomesfrom transfected cell supernatant. Next, El-Andaloussi et al. detailcrucial steps for loading RNA into exosomes. Finally, El-Andaloussi etal. outline how to use exosomes to efficiently deliver RNA in vitro andin vivo in mouse brain. Examples of anticipated results in whichexosome-mediated RNA delivery is evaluated by functional assays andimaging are also provided. The entire protocol takes ˜3 weeks. Deliveryor administration according to the invention may be performed usingexosomes produced from self-derived dendritic cells. From the hereinteachings, this can be employed in the practice of the invention

In another embodiment, the plasma exosomes of Wahlgren et al. (NucleicAcids Research, 2012, Vol. 40, No. 17 e130) are contemplated. Exosomesare nano-sized vesicles (30-90 nm in size) produced by many cell types,including dendritic cells (DC), B cells, T cells, mast cells, epithelialcells and tumor cells. These vesicles are formed by inward budding oflate endosomes and are then released to the extracellular environmentupon fusion with the plasma membrane. Because exosomes naturally carryRNA between cells, this property may be useful in gene therapy, and fromthis disclosure can be employed in the practice of the instantinvention.

Exosomes from plasma can be prepared by centrifugation of buffy coat at900 g for 20 min to isolate the plasma followed by harvesting cellsupernatants, centrifuging at 300 g for 10 min to eliminate cells and at16 500 g for 30 min followed by filtration through a 0.22 mm filter.Exosomes are pelleted by ultracentrifugation at 120 000 g for 70 min.Chemical transfection of siRNA into exosomes is carried out according tothe manufacturer's instructions in RNAi Human/Mouse Starter Kit(Quiagen, Hilden, Germany). siRNA is added to 100 ml PBS at a finalconcentration of 2 mmol/ml. After adding HiPerFect transfection reagent,the mixture is incubated for 10 min at RT. In order to remove the excessof micelles, the exosomes are re-isolated using aldehyde/sulfate latexbeads. The chemical transfection of nucleic acid-targeting system intoexosomes may be conducted similarly to siRNA. The exosomes may beco-cultured with monocytes and lymphocytes isolated from the peripheralblood of healthy donors. Therefore, it may be contemplated that exosomescontaining nucleic acid-targeting system may be introduced to monocytesand lymphocytes of and autologously reintroduced into a human.Accordingly, delivery or administration according to the invention maybe performed using plasma exosomes.

Liposomes

Delivery or administration according to the invention can be performedwith liposomes. Liposomes are spherical vesicle structures composed of auni- or multilamellar lipid bilayer surrounding internal aqueouscompartments and a relatively impermeable outer lipophilic phospholipidbilayer. Liposomes have gained considerable attention as drug deliverycarriers because they are biocompatible, nontoxic, can deliver bothhydrophilic and lipophilic drug molecules, protect their cargo fromdegradation by plasma enzymes, and transport their load acrossbiological membranes and the blood brain barrier (BBB) (see, e.g., Spuchand Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12pages, 2011. doi:10.1155/2011/469679 for review).

Liposomes can be made from several different types of lipids; however,phospholipids are most commonly used to generate liposomes as drugcarriers. Although liposome formation is spontaneous when a lipid filmis mixed with an aqueous solution, it can also be expedited by applyingforce in the form of shaking by using a homogenizer, sonicator, or anextrusion apparatus (see, e.g., Spuch and Navarro, Journal of DrugDelivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679 for review).

Several other additives may be added to liposomes in order to modifytheir structure and properties. For instance, either cholesterol orsphingomyelin may be added to the liposomal mixture in order to helpstabilize the liposomal structure and to prevent the leakage of theliposomal inner cargo. Further, liposomes are prepared from hydrogenatedegg phosphatidylcholine or egg phosphatidylcholine, cholesterol, anddicetyl phosphate, and their mean vesicle sizes were adjusted to about50 and 100 nm. (see, e.g., Spuch and Navarro, Journal of Drug Delivery,vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679for review).

A liposome formulation may be mainly comprised of natural phospholipidsand lipids such as 1,2-distearoryl-sn-glycero-3-phosphatidyl choline(DSPC), sphingomyelin, egg phosphatidylcholines andmonosialoganglioside. Since this formulation is made up of phospholipidsonly, liposomal formulations have encountered many challenges, one ofthe ones being the instability in plasma. Several attempts to overcomethese challenges have been made, specifically in the manipulation of thelipid membrane. One of these attempts focused on the manipulation ofcholesterol. Addition of cholesterol to conventional formulationsreduces rapid release of the encapsulated bioactive compound into theplasma or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increasesthe stability (see, e.g., Spuch and Navarro, Journal of Drug Delivery,vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679for review).

In a particularly advantageous embodiment, Trojan Horse liposomes (alsoknown as Molecular Trojan Horses) are desirable and protocols may befound at http://cshprotocols.cshlp.org/content/2010/4/pdb.prot5407.long.These particles allow delivery of a transgene to the entire brain afteran intravascular injection. Without being bound by limitation, it isbelieved that neutral lipid particles with specific antibodiesconjugated to surface allow crossing of the blood brain barrier viaendocytosis. Applicant postulates utilizing Trojan Horse Liposomes todeliver the CRISPR family of nucleases to the brain via an intravascularinjection, which would allow whole brain transgenic animals without theneed for embryonic manipulation. About 1-5 g of DNA or RNA may becontemplated for in vivo administration in liposomes.

In another embodiment, the nucleic acid-targeting system or componentsthereof may be administered in liposomes, such as a stablenucleic-acid-lipid particle (SNALP) (see, e.g., Morrissey et al., NatureBiotechnology, Vol. 23, No. 8, August 2005). Daily intravenousinjections of about 1, 3 or 5 mg/kg/day of a specific nucleicacid-targeting system targeted in a SNALP are contemplated. The dailytreatment may be over about three days and then weekly for about fiveweeks. In another embodiment, a specific nucleic acid-targeting systemencapsulated SNALP) administered by intravenous injection to at doses ofabout 1 or 2.5 mg/kg are also contemplated (see, e.g., Zimmerman et al.,Nature Letters, Vol. 441, 4 May 2006). The SNALP formulation may containthe lipids 3-N-[(wmethoxypoly(ethylene glycol) 2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA),1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, in a2:40:10:48 molar percent ratio (see, e.g., Zimmerman et al., NatureLetters, Vol. 441, 4 May 2006).

In another embodiment, stable nucleic-acid-lipid particles (SNALPs) haveproven to be effective delivery molecules to highly vascularizedHepG2-derived liver tumors but not in poorly vascularized HCT-116derived liver tumors (see, e.g., Li, Gene Therapy (2012) 19, 775-780).The SNALP liposomes may be prepared by formulating D-Lin-DMA andPEG-C-DMA with distearoylphosphatidylcholine (DSPC), Cholesterol andsiRNA using a 25:1 lipid/siRNA ratio and a 48/40/10/2 molar ratio ofCholesterol/D-Lin-DMA/DSPC/PEG-C-DMA. The resulted SNALP liposomes areabout 80-100 nm in size.

In yet another embodiment, a SNALP may comprise synthetic cholesterol(Sigma-Aldrich, St Louis, Mo., USA), dipalmitoylphosphatidylcholine(Avanti Polar Lipids, Alabaster, Ala., USA), 3-N-[(w-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyrestyloxypropylamine, andcationic 1,2-dilinoleyloxy-3-N,Ndimethylaminopropane (see, e.g.,Geisbert et al., Lancet 2010; 375: 1896-905). A dosage of about 2 mg/kgtotal nucleic acid-targeting systemper dose administered as, forexample, a bolus intravenous infusion may be contemplated.

In yet another embodiment, a SNALP may comprise synthetic cholesterol(Sigma-Aldrich), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC;Avanti Polar Lipids Inc.), PEG-cDMA, and1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA) (see, e.g.,Judge, J. Clin. Invest. 119:661-673 (2009)). Formulations used for invivo studies may comprise a final lipid/RNA mass ratio of about 9:1.

The safety profile of RNAi nanomedicines has been reviewed by Barros andGollob of Alnylam Pharmaceuticals (see, e.g., Advanced Drug DeliveryReviews 64 (2012) 1730-1737). The stable nucleic acid lipid particle(SNALP) is comprised of four different lipids—an ionizable lipid(DLinDMA) that is cationic at low pH, a neutral helper lipid,cholesterol, and a diffusible polyethylene glycol (PEG)-lipid. Theparticle is approximately 80 nm in diameter and is charge-neutral atphysiologic pH. During formulation, the ionizable lipid serves tocondense lipid with the anionic RNA during particle formation. Whenpositively charged under increasingly acidic endosomal conditions, theionizable lipid also mediates the fusion of SNALP with the endosomalmembrane enabling release of RNA into the cytoplasm. The PEG-lipidstabilizes the particle and reduces aggregation during formulation, andsubsequently provides a neutral hydrophilic exterior that improvespharmacokinetic properties.

To date, two clinical programs have been initiated using SNALPformulations with RNA. Tekmira Pharmaceuticals recently completed aphase I single-dose study of SNALP-ApoB in adult volunteers withelevated LDL cholesterol. ApoB is predominantly expressed in the liverand jejunum and is essential for the assembly and secretion of VLDL andLDL. Seventeen subjects received a single dose of SNALP-ApoB (doseescalation across 7 dose levels). There was no evidence of livertoxicity (anticipated as the potential dose-limiting toxicity based onpreclinical studies). One (of two) subjects at the highest doseexperienced flu-like symptoms consistent with immune system stimulation,and the decision was made to conclude the trial.

Alnylam Pharmaceuticals has similarly advanced ALN-TTR01, which employsthe SNALP technology described above and targets hepatocyte productionof both mutant and wild-type TTR to treat TTR amyloidosis (ATTR). ThreeATTR syndromes have been described: familial amyloidotic polyneuropathy(FAP) and familial amyloidotic cardiomyopathy (FAC)-both caused byautosomal dominant mutations in TTR; and senile systemic amyloidosis(SSA) cause by wildtype TTR. A placebo-controlled, singledose-escalation phase I trial of ALN-TTR0 1 was recently completed inpatients with ATTR. ALN-TTR01 was administered as a 15-minute IVinfusion to 31 patients (23 with study drug and 8 with placebo) within adose range of 0.01 to 1.0 mg/kg (based on siRNA). Treatment was welltolerated with no significant increases in liver function tests.Infusion-related reactions were noted in 3 of 23 patients at ≥0.4 mg/kg;all responded to slowing of the infusion rate and all continued onstudy. Minimal and transient elevations of serum cytokines IL-6, IP-10and IL-Ira were noted in two patients at the highest dose of 1 mg/kg (asanticipated from preclinical and NHP studies). Lowering of serum TTR,the expected pharmacodynamics effect of ALN-TTR01, was observed at 1mg/kg.

In yet another embodiment, a SNALP may be made by solubilizing acationic lipid, DSPC, cholesterol and PEG-lipid e.g., in ethanol, e.g.,at a molar ratio of 40:10:40:10, respectively (see, Semple et al.,Nature Niotechnology, Volume 28 Number 2 Feb. 2010, pp. 172-177). Thelipid mixture was added to an aqueous buffer (50 mM citrate, pH 4) withmixing to a final ethanol and lipid concentration of 30% (vol/vol) and6.1 mg/ml, respectively, and allowed to equilibrate at 22° C. for 2 minbefore extrusion. The hydrated lipids were extruded through two stacked80 nm pore-sized filters (Nuclepore) at 22° C. using a Lipex Extruder(Northern Lipids) until a vesicle diameter of 70-90 nm, as determined bydynamic light scattering analysis, was obtained. This generally required1-3 passes. The siRNA (solubilized in a 50 mM citrate, pH 4 aqueoussolution containing 30% ethanol) was added to the pre-equilibrated (35°C.) vesicles at a rate of ˜5 ml/min with mixing. After a final targetsiRNA/lipid ratio of 0.06 (wt/wt) was reached, the mixture was incubatedfor a further 30 min at 35° C. to allow vesicle reorganization andencapsulation of the siRNA. The ethanol was then removed and theexternal buffer replaced with PBS (155 mM NaCl, 3 mM Na₂HPO₄, 1 mMKH₂PO₄, pH 7.5) by either dialysis or tangential flow diafiltration.siRNA were encapsulated in SNALP using a controlled step-wise dilutionmethod process. The lipid constituents of KC2-SNALP were DLin-KC2-DMA(cationic lipid), dipalmitoylphosphatidylcholine (DPPC; Avanti PolarLipids), synthetic cholesterol (Sigma) and PEG-C-DMA used at a molarratio of 57.1:7.1:34.3:1.4. Upon formation of the loaded particles,SNALP were dialyzed against PBS and filter sterilized through a 0.2 μmfilter before use. Mean particle sizes were 75-85 nm and 90-95% of thesiRNA was encapsulated within the lipid particles. The final siRNA/lipidratio in formulations used for in vivo testing was ˜0.15 (wt/wt).LNP-siRNA systems containing Factor VII siRNA were diluted to theappropriate concentrations in sterile PBS immediately before use and theformulations were administered intravenously through the lateral tailvein in a total volume of 10 ml/kg. This method and these deliverysystems may be extrapolated to the nucleic acid-targeting system of thepresent invention.

Other Lipids

Other cationic lipids, such as amino lipid2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) maybe utilized to encapsulate nucleic acid-targeting system or componentsthereof or nucleic acid molecule(s) coding therefor e.g., similar toSiRNA (see, e.g., Jayaraman, Angew. Chem. Int. Ed. 2012, 51, 8529-8533),and hence may be employed in the practice of the invention. A preformedvesicle with the following lipid composition may be contemplated: aminolipid, distearoylphosphatidylcholine (DSPC), cholesterol and(R)-2,3-bis(octadecyloxy) propyl-1-(methoxy poly(ethyleneglycol)2000)propylcarbamate (PEG-lipid) in the molar ratio 40/10/40/10,respectively, and a FVII siRNA/total lipid ratio of approximately 0.05(w/w). To ensure a narrow particle size distribution in the range of70-90 nm and a low polydispersity index of 0.11±0.04 (n=56), theparticles may be extruded up to three times through 80 nm membranesprior to adding the guide RNA. Particles containing the highly potentamino lipid 16 may be used, in which the molar ratio of the four lipidcomponents 16, DSPC, cholesterol and PEG-lipid (50/10/38.5/1.5) whichmay be further optimized to enhance in vivo activity.

Michael S D Kormann et al. (“Expression of therapeutic proteins afterdelivery of chemically modified mRNA in mice: Nature Biotechnology,Volume:29, Pages: 154-157 (2011)) describes the use of lipid envelopesto deliver RNA. Use of lipid envelopes is also preferred in the presentinvention.

In another embodiment, lipids may be formulated with the nucleicacid-targeting system of the present invention or component(s) thereofor nucleic acid molecule(s) coding therefor to form lipid nanoparticles(LNPs). Lipids include, but are not limited to, DLin-KC2-DMA4, C12-200and colipids disteroylphosphatidyl choline, cholesterol, and PEG-DMG maybe formulated with RNA-targeting system instead of siRNA (see, e.g.,Novobrantseva, Molecular Therapy-Nucleic Acids (2012) 1, e4;doi:10.1038/mtna.2011.3) using a spontaneous vesicle formationprocedure. The component molar ratio may be about 50/10/38.5/1.5(DLin-KC2-DMA or C12-200/disteroylphosphatidylcholine/cholesterol/PEG-DMG). The final lipid:siRNA weight ratio may be˜12:1 and 9:1 in the case of DLin-KC2-DMA and C12-200 lipid particles(LNPs), respectively. The formulations may have mean particle diametersof ˜80 nm with >90% entrapment efficiency. A 3 mg/kg dose may becontemplated.

Tekmira has a portfolio of approximately 95 patent families, in the U.S.and abroad, that are directed to various aspects of LNPs and LNPformulations (see, e.g., U.S. Pat. Nos. 7,982,027; 7,799,565; 8,058,069;8,283,333; 7,901,708; 7,745,651; 7,803,397; 8,101,741; 8,188,263;7,915,399; 8,236,943 and 7,838,658 and European Pat. Nos 1766035;1519714; 1781593 and 1664316), all of which may be used and/or adaptedto the present invention.

The nucleic acid-targetingsystem or components thereof or nucleic acidmolecule(s) coding therefor may be delivered encapsulated in PLGAMicrospheres such as that further described in US published applications20130252281 and 20130245107 and 20130244279 (assigned to ModernaTherapeutics) which relate to aspects of formulation of compositionscomprising modified nucleic acid molecules which may encode a protein, aprotein precursor, or a partially or fully processed form of the proteinor a protein precursor. The formulation may have a molar ratio50:10:38.5:1.5-3.0 (cationic lipid:fusogenic lipid:cholesterol:PEGlipid). The PEG lipid may be selected from, but is not limited toPEG-c-DOMG, PEG-DMG. The fusogenic lipid may be DSPC. See also, Schrumet al., Delivery and Formulation of Engineered Nucleic Acids, USpublished application 20120251618.

Nanomerics' technology addresses bioavailability challenges for a broadrange of therapeutics, including low molecular weight hydrophobic drugs,peptides, and nucleic acid based therapeutics (plasmid, siRNA, miRNA).Specific administration routes for which the technology has demonstratedclear advantages include the oral route, transport across theblood-brain-barrier, delivery to solid tumours, as well as to the eye.See, e.g., Mazza et al., 2013, ACS Nano. 2013 Feb. 26; 7(2):1016-26;Uchegbu and Siew, 2013, J Pharm Sci. 102(2):305-10 and Lalatsa et al.,2012, J Control Release. 2012 Jul. 20; 161(2):523-36.

US Patent Publication No. 20050019923 describes cationic dendrimers fordelivering bioactive molecules, such as polynucleotide molecules,peptides and polypeptides and/or pharmaceutical agents, to a mammalianbody. The dendrimers are suitable for targeting the delivery of thebioactive molecules to, for example, the liver, spleen, lung, kidney orheart (or even the brain). Dendrimers are synthetic 3-dimensionalmacromolecules that are prepared in a step-wise fashion from simplebranched monomer units, the nature and functionality of which can beeasily controlled and varied. Dendrimers are synthesized from therepeated addition of building blocks to a multifunctional core(divergent approach to synthesis), or towards a multifunctional core(convergent approach to synthesis) and each addition of a 3-dimensionalshell of building blocks leads to the formation of a higher generationof the dendrimers. Polypropylenimine dendrimers start from adiaminobutane core to which is added twice the number of amino groups bya double Michael addition of acrylonitrile to the primary aminesfollowed by the hydrogenation of the nitriles. This results in adoubling of the amino groups. Polypropylenimine dendrimers contain 100%protonable nitrogens and up to 64 terminal amino groups (generation 5,DAB 64). Protonable groups are usually amine groups which are able toaccept protons at neutral pH. The use of dendrimers as gene deliveryagents has largely focused on the use of the polyamidoamine. andphosphorous containing compounds with a mixture of amine/amide orN—P(O₂)S as the conjugating units respectively with no work beingreported on the use of the lower generation polypropylenimine dendrimersfor gene delivery. Polypropylenimine dendrimers have also been studiedas pH sensitive controlled release systems for drug delivery and fortheir encapsulation of guest molecules when chemically modified byperipheral amino acid groups. The cytotoxicity and interaction ofpolypropylenimine dendrimers with DNA as well as the transfectionefficacy of DAB 64 has also been studied.

US Patent Publication No. 20050019923 is based upon the observationthat, contrary to earlier reports, cationic dendrimers, such aspolypropylenimine dendrimers, display suitable properties, such asspecific targeting and low toxicity, for use in the targeted delivery ofbioactive molecules, such as genetic material. In addition, derivativesof the cationic dendrimer also display suitable properties for thetargeted delivery of bioactive molecules. See also, Bioactive Polymers,US published application 20080267903, which discloses “Various polymers,including cationic polyamine polymers and dendrimeric polymers, areshown to possess anti-proliferative activity, and may therefore beuseful for treatment of disorders characterised by undesirable cellularproliferation such as neoplasms and tumours, inflammatory disorders(including autoimmune disorders), psoriasis and atherosclerosis. Thepolymers may be used alone as active agents, or as delivery vehicles forother therapeutic agents, such as drug molecules or nucleic acids forgene therapy. In such cases, the polymers' own intrinsic anti-tumouractivity may complement the activity of the agent to be delivered.” Thedisclosures of these patent publications may be employed in conjunctionwith herein teachings for delivery of nucleic acid-targetingsystem(s) orcomponent(s) thereof or nucleic acid molecule(s) coding therefor.

Supercharged Proteins

Supercharged proteins are a class of engineered or naturally occurringproteins with unusually high positive or negative net theoretical chargeand may be employed in delivery of nucleic acid-targetingsystem(s) orcomponent(s) thereof or nucleic acid molecule(s) coding therefor. Bothsupernegatively and superpositively charged proteins exhibit aremarkable ability to withstand thermally or chemically inducedaggregation. Superpositively charged proteins are also able to penetratemammalian cells. Associating cargo with these proteins, such as plasmidDNA, RNA, or other proteins, can enable the functional delivery of thesemacromolecules into mammalian cells both in vitro and in vivo. DavidLiu's lab reported the creation and characterization of superchargedproteins in 2007 (Lawrence et al., 2007, Journal of the AmericanChemical Society 129, 10110-10112).

The nonviral delivery of RNA and plasmid DNA into mammalian cells arevaluable both for research and therapeutic applications (Akinc et al.,2010, Nat. Biotech. 26, 561-569). Purified +36 GFP protein (or othersuperpositively charged protein) is mixed with RNAs in the appropriateserum-free media and allowed to complex prior addition to cells.Inclusion of serum at this stage inhibits formation of the superchargedprotein-RNA complexes and reduces the effectiveness of the treatment.The following protocol has been found to be effective for a variety ofcell lines (McNaughton et al., 2009, Proc. Natl. Acad. Sci. USA 106,6111-6116). However, pilot experiments varying the dose of protein andRNA should be performed to optimize the procedure for specific celllines.

(1) One day before treatment, plate 1×10⁵ cells per well in a 48-wellplate.

(2) On the day of treatment, dilute purified +36 GFP protein inserumfree media to a final concentration 200 nM. Add RNA to a finalconcentration of 50 nM. Vortex to mix and incubate at room temperaturefor 10 min.

(3) During incubation, aspirate media from cells and wash once with PBS.

(4) Following incubation of +36 GFP and RNA, add the protein-RNAcomplexes to cells.

(5) Incubate cells with complexes at 37° C. for 4 h.

(6) Following incubation, aspirate the media and wash three times with20 U/mL heparin PBS. Incubate cells with serum-containing media for afurther 48 h or longer depending upon the assay for activity.

(7) Analyze cells by immunoblot, qPCR, phenotypic assay, or otherappropriate method.

David Liu's lab has further found +36 GFP to be an effective plasmiddelivery reagent in a range of cells. As plasmid DNA is a larger cargothan siRNA, proportionately more +36 GFP protein is required toeffectively complex plasmids. For effective plasmid delivery Applicantshave developed a variant of +36 GFP bearing a C-terminal HA2 peptidetag, a known endosome-disrupting peptide derived from the influenzavirus hemagglutinin protein. The following protocol has been effectivein a variety of cells, but as above it is advised that plasmid DNA andsupercharged protein doses be optimized for specific cell lines anddelivery applications.

(1) One day before treatment, plate 1×10⁵ per well in a 48-well plate.

(2) On the day of treatment, dilute purified

36 GFP protein in serumfree media to a final concentration 2 mM. Add 1mg of plasmid DNA. Vortex to mix and incubate at room temperature for 10min.

(3) During incubation, aspirate media from cells and wash once with PBS.

(4) Following incubation of

36 GFP and plasmid DNA, gently add the protein-DNA complexes to cells.

(5) Incubate cells with complexes at 37 C for 4 h.

(6) Following incubation, aspirate the media and wash with PBS. Incubatecells in serum-containing media and incubate for a further 24-48 h.

(7) Analyze plasmid delivery (e.g., by plasmid-driven gene expression)as appropriate.

See also, e.g., McNaughton et al., Proc. Natl. Acad. Sci. USA 106,6111-6116 (2009); Cronican et al., ACS Chemical Biology 5, 747-752(2010); Cronican et al., Chemistry & Biology 18, 833-838 (2011);Thompson et al., Methods in Enzymology 503, 293-319 (2012); Thompson, D.B., et al., Chemistry & Biology 19 (7), 831-843 (2012). The methods ofthe super charged proteins may be used and/or adapted for delivery ofthe nucleic acid-targeting system of the present invention. Thesesystems of Dr. Lui and documents herein in conjunction with hereinteachings can be employed in the delivery of nucleic acid-targetingsystem(s) or component(s) thereof or nucleic acid molecule(s) codingtherefor.

Cell Penetrating Peptides (CPPs)

In yet another embodiment, cell penetrating peptides (CPPs) arecontemplated for the delivery of the CRISPR Cas system. CPPs are shortpeptides that facilitate cellular uptake of various molecular cargo(from nanosize particles to small chemical molecules and large fragmentsof DNA). The term “cargo” as used herein includes but is not limited tothe group consisting of therapeutic agents, diagnostic probes, peptides,nucleic acids, antisense oligonucleotides, plasmids, proteins, particlesincluding nanoparticles, liposomes, chromophores, small molecules andradioactive materials. In aspects of the invention, the cargo may alsocomprise any component of the CRISPR Cas system or the entire functionalCRISPR Cas system. Aspects of the present invention further providemethods for delivering a desired cargo into a subject comprising: (a)preparing a complex comprising the cell penetrating peptide of thepresent invention and a desired cargo, and (b) orally, intraarticularly,intraperitoneally, intrathecally, intrarterially, intranasally,intraparenchymally, subcutaneously, intramuscularly, intravenously,dermally, intrarectally, or topically administering the complex to asubject. The cargo is associated with the peptides either throughchemical linkage via covalent bonds or through non-covalentinteractions.

The function of the CPPs are to deliver the cargo into cells, a processthat commonly occurs through endocytosis with the cargo delivered to theendosomes of living mammalian cells. Cell-penetrating peptides are ofdifferent sizes, amino acid sequences, and charges but all CPPs have onedistinct characteristic, which is the ability to translocate the plasmamembrane and facilitate the delivery of various molecular cargoes to thecytoplasm or an organelle. CPP translocation may be classified intothree main entry mechanisms: direct penetration in the membrane,endocytosis-mediated entry, and translocation through the formation of atransitory structure. CPPs have found numerous applications in medicineas drug delivery agents in the treatment of different diseases includingcancer and virus inhibitors, as well as contrast agents for celllabeling. Examples of the latter include acting as a carrier for GFP,MRI contrast agents, or quantum dots. CPPs hold great potential as invitro and in vivo delivery vectors for use in research and medicine.CPPs typically have an amino acid composition that either contains ahigh relative abundance of positively charged amino acids such as lysineor arginine or has sequences that contain an alternating pattern ofpolar/charged amino acids and non-polar, hydrophobic amino acids. Thesetwo types of structures are referred to as polycationic or amphipathic,respectively. A third class of CPPs are the hydrophobic peptides,containing only apolar residues, with low net charge or have hydrophobicamino acid groups that are crucial for cellular uptake. One of theinitial CPPs discovered was the trans-activating transcriptionalactivator (Tat) from Human Immunodeficiency Virus 1 (HIV-1) which wasfound to be efficiently taken up from the surrounding media by numerouscell types in culture. Since then, the number of known CPPs has expandedconsiderably and small molecule synthetic analogues with more effectiveprotein transduction properties have been generated. CPPs include butare not limited to Penetratin, Tat (48-60), Transportan, and (R-AhX-R4)(Ahx=aminohexanoyl).

U.S. Pat. No. 8,372,951, provides a CPP derived from eosinophil cationicprotein (ECP) which exhibits highly cell-penetrating efficiency and lowtoxicity. Aspects of delivering the CPP with its cargo into a vertebratesubject are also provided. Further aspects of CPPs and their deliveryare described in U.S. Pat. Nos. 8,575,305; 8,614,194 and 8,044,019. CPPscan be used to deliver the CRISPR-Cas system or components thereof. ThatCPPs can be employed to deliver the CRISPR-Cas system or componentsthereof is also provided in the manuscript “Gene disruption bycell-penetrating peptide-mediated delivery of Cas9 protein and guideRNA”, by Suresh Ramakrishna, Abu-Bonsrah Kwaku Dad, Jagadish Beloor, etal. Genome Res. 2014 Apr. 2. [Epub ahead of print], incorporated byreference in its entirety, wherein it is demonstrated that treatmentwith CPP-conjugated recombinant Cas9 protein and CPP-complexed guideRNAs lead to endogenous gene disruptions in human cell lines. In thepaper the Cas9 protein was conjugated to CPP via a thioether bond,whereas the guide RNA was complexed with CPP, forming condensed,positively charged particles. It was shown that simultaneous andsequential treatment of human cells, including embryonic stem cells,dermal fibroblasts, HEK293T cells, HeLa cells, and embryonic carcinomacells, with the modified Cas9 and guide RNA led to efficient genedisruptions with reduced off-target mutations relative to plasmidtransfections.

Implantable Devices

In another embodiment, implantable devices are also contemplated fordelivery of the nucleic acid-targeting system or component(s) thereof ornucleic acid molecule(s) coding therefor. For example, US PatentPublication 20110195123 discloses an implantable medical device whichelutes a drug locally and in prolonged period is provided, includingseveral types of such a device, the treatment modes of implementationand methods of implantation. The device comprising of polymericsubstrate, such as a matrix for example, that is used as the devicebody, and drugs, and in some cases additional scaffolding materials,such as metals or additional polymers, and materials to enhancevisibility and imaging. An implantable delivery device can beadvantageous in providing release locally and over a prolonged period,where drug is released directly to the extracellular matrix (ECM) of thediseased area such as tumor, inflammation, degeneration or forsymptomatic objectives, or to injured smooth muscle cells, or forprevention. One kind of drug is RNA, as disclosed above, and this systemmay be used/and or adapted to the nucleic acid-targeting system of thepresent invention. The modes of implantation in some embodiments areexisting implantation procedures that are developed and used today forother treatments, including brachytherapy and needle biopsy. In suchcases the dimensions of the new implant described in this invention aresimilar to the original implant. Typically a few devices are implantedduring the same treatment procedure.

US Patent Publication 20110195123, provides a drug delivery implantableor insertable system, including systems applicable to a cavity such asthe abdominal cavity and/or any other type of administration in whichthe drug delivery system is not anchored or attached, comprising abiostable and/or degradable and/or bioabsorbable polymeric substrate,which may for example optionally be a matrix. It should be noted thatthe term “insertion” also includes implantation. The drug deliverysystem is preferably implemented as a “Loder” as described in US PatentPublication 20110195123.

The polymer or plurality of polymers are biocompatible, incorporating anagent and/or plurality of agents, enabling the release of agent at acontrolled rate, wherein the total volume of the polymeric substrate,such as a matrix for example, in some embodiments is optionally andpreferably no greater than a maximum volume that permits a therapeuticlevel of the agent to be reached. As a non-limiting example, such avolume is preferably within the range of 0.1 m³ to 1000 mm³, as requiredby the volume for the agent load. The Loder may optionally be larger,for example when incorporated with a device whose size is determined byfunctionality, for example and without limitation, a knee joint, anintra-uterine or cervical ring and the like.

The drug delivery system (for delivering the composition) is designed insome embodiments to preferably employ degradable polymers, wherein themain release mechanism is bulk erosion; or in some embodiments, nondegradable, or slowly degraded polymers are used, wherein the mainrelease mechanism is diffusion rather than bulk erosion, so that theouter part functions as membrane, and its internal part functions as adrug reservoir, which practically is not affected by the surroundingsfor an extended period (for example from about a week to about a fewmonths). Combinations of different polymers with different releasemechanisms may also optionally be used. The concentration gradient atthe surface is preferably maintained effectively constant during asignificant period of the total drug releasing period, and therefore thediffusion rate is effectively constant (termed “zero mode” diffusion).By the term “constant” it is meant a diffusion rate that is preferablymaintained above the lower threshold of therapeutic effectiveness, butwhich may still optionally feature an initial burst and/or mayfluctuate, for example increasing and decreasing to a certain degree.The diffusion rate is preferably so maintained for a prolonged period,and it can be considered constant to a certain level to optimize thetherapeutically effective period, for example the effective silencingperiod.

The drug delivery system optionally and preferably is designed to shieldthe nucleotide based therapeutic agent from degradation, whetherchemical in nature or due to attack from enzymes and other factors inthe body of the subject.

The drug delivery system of US Patent Publication 20110195123 isoptionally associated with sensing and/or activation appliances that areoperated at and/or after implantation of the device, by non and/orminimally invasive methods of activation and/oracceleration/deceleration, for example optionally including but notlimited to thermal heating and cooling, laser beams, and ultrasonic,including focused ultrasound and/or RF (radiofrequency) methods ordevices.

According to some embodiments of US Patent Publication 20110195123, thesite for local delivery may optionally include target sitescharacterized by high abnormal proliferation of cells, and suppressedapoptosis, including tumors, active and or chronic inflammation andinfection including autoimmune diseases states, degenerating tissueincluding muscle and nervous tissue, chronic pain, degenerative sites,and location of bone fractures and other wound locations for enhancementof regeneration of tissue, and injured cardiac, smooth and striatedmuscle.

The site for implantation of the composition, or target site, preferablyfeatures a radius, area and/or volume that is sufficiently small fortargeted local delivery. For example, the target site optionally has adiameter in a range of from about 0.1 mm to about 5 cm.

The location of the target site is preferably selected for maximumtherapeutic efficacy. For example, the composition of the drug deliverysystem (optionally with a device for implantation as described above) isoptionally and preferably implanted within or in the proximity of atumor environment, or the blood supply associated thereof.

For example the composition (optionally with the device) is optionallyimplanted within or in the proximity to pancreas, prostate, breast,liver, via the nipple, within the vascular system and so forth.

The target location is optionally selected from the group comprising,consisting essentially of, or consisting of (as non-limiting examplesonly, as optionally any site within the body may be suitable forimplanting a Loder): 1. brain at degenerative sites like in Parkinson orAlzheimer disease at the basal ganglia, white and gray matter; 2. spineas in the case of amyotrophic lateral sclerosis (ALS); 3. uterine cervixto prevent HPV infection; 4. active and chronic inflammatory joints; 5.dermis as in the case of psoriasis; 6. sympathetic and sensoric nervoussites for analgesic effect; 7. Intra osseous implantation; 8. acute andchronic infection sites; 9. Intra vaginal; 10. Inner ear-auditorysystem, labyrinth of the inner ear, vestibular system; 11. Intratracheal; 12. Intra-cardiac; coronary, epicardiac; 13. urinary bladder;14. biliary system; 15. parenchymal tissue including and not limited tothe kidney, liver, spleen; 16. lymph nodes; 17. salivary glands; 18.dental gums; 19. Intra-articular (into joints); 20. Intra-ocular; 21.Brain tissue; 22. Brain ventricles; 23. Cavities, including abdominalcavity (for example but without limitation, for ovary cancer); 24. Intraesophageal and 25. Intra rectal.

Optionally insertion of the system (for example a device containing thecomposition) is associated with injection of material to the ECM at thetarget site and the vicinity of that site to affect local pH and/ortemperature and/or other biological factors affecting the diffusion ofthe drug and/or drug kinetics in the ECM, of the target site and thevicinity of such a site.

Optionally, according to some embodiments, the release of said agentcould be associated with sensing and/or activation appliances that areoperated prior and/or at and/or after insertion, by non and/or minimallyinvasive and/or else methods of activation and/oracceleration/deceleration, including laser beam, radiation, thermalheating and cooling, and ultrasonic, including focused ultrasound and/orRF (radiofrequency) methods or devices, and chemical activators.

According to other embodiments of U.S. Patent Publication 20110195123,the drug preferably comprises a RNA, for example for localized cancercases in breast, pancreas, brain, kidney, bladder, lung, and prostate asdescribed below. Although exemplified with RNAi, many drugs areapplicable to be encapsulated in Loder, and can be used in associationwith this invention, as long as such drugs can be encapsulated with theLoder substrate, such as a matrix for example, and this system may beused and/or adapted to deliver the nucleic acid-targeting system of thepresent invention.

As another example of a specific application, neuro and musculardegenerative diseases develop due to abnormal gene expression. Localdelivery of RNAs may have therapeutic properties for interfering withsuch abnormal gene expression. Local delivery of anti apoptotic, antiinflammatory and anti degenerative drugs including small drugs andmacromolecules may also optionally be therapeutic. In such cases theLoder is applied for prolonged release at constant rate and/or through adedicated device that is implanted separately. All of this may be usedand/or adapted to the nucleic acid-targeting system of the presentinvention.

As yet another example of a specific application, psychiatric andcognitive disorders are treated with gene modifiers. Gene knockdown is atreatment option. Loders locally delivering agents to central nervoussystem sites are therapeutic options for psychiatric and cognitivedisorders including but not limited to psychosis, bi-polar diseases,neurotic disorders and behavioral maladies. The Loders could alsodeliver locally drugs including small drugs and macromolecules uponimplantation at specific brain sites. All of this may be used and/oradapted to the nucleic acid-targeting system of the present invention.

As another example of a specific application, silencing of innate and/oradaptive immune mediators at local sites enables the prevention of organtransplant rejection. Local delivery of RNAs and immunomodulatingreagents with the Loder implanted into the transplanted organ and/or theimplanted site renders local immune suppression by repelling immunecells such as CD8 activated against the transplanted organ. All of thismay be used/and or adapted to the nucleic acid-targeting system of thepresent invention.

As another example of a specific application, vascular growth factorsincluding VEGFs and angiogenin and others are essential forneovascularization. Local delivery of the factors, peptides,peptidomimetics, or suppressing their repressors is an importanttherapeutic modality; silencing the repressors and local delivery of thefactors, peptides, macromolecules and small drugs stimulatingangiogenesis with the Loder is therapeutic for peripheral, systemic andcardiac vascular disease.

The method of insertion, such as implantation, may optionally already beused for other types of tissue implantation and/or for insertions and/orfor sampling tissues, optionally without modifications, or alternativelyoptionally only with non-major modifications in such methods. Suchmethods optionally include but are not limited to brachytherapy methods,biopsy, endoscopy with and/or without ultrasound, such as ERCP,stereotactic methods into the brain tissue, Laparoscopy, includingimplantation with a laparoscope into joints, abdominal organs, thebladder wall and body cavities.

Implantable device technology herein discussed can be employed withherein teachings and hence by this disclosure and the knowledge in theart, CRISPR-Cas system or components thereof or nucleic acid moleculesthereof or encoding or providing components may be delivered via animplantable device.

Patient-Specific Screening Methods

A nucleic acid-targeting system that targets RNA, e.g., trinucleotiderepeats can be used to screen patients or patent samples for thepresence of such repeats. The repeats can be the target of the RNA ofthe nucleic acid-targeting system, and if there is binding thereto bythe nucleic acid-targeting system, that binding can be detected, tothereby indicate that such a repeat is present. Thus, a nucleicacid-targeting system can be used to screen patients or patient samplesfor the presence of the repeat. The patient can then be administeredsuitable compound(s) to address the condition; or, can be administered anucleic acid-targeting system to bind to and cause insertion, deletionor mutation and alleviate the condition.

The invention uses nucleic acids to bind target RNA sequences.

CRISPR Effector Protein mRNA and Guide RNA

CRISPR effector protein mRNA and guide RNA might also be deliveredseparately. CRISPR effector protein mRNA can be delivered prior to theguide RNA to give time for CRISPR effector protein to be expressed.CRISPR effector protein mRNA might be administered 1-12 hours(preferably around 2-6 hours) prior to the administration of guide RNA.

Alternatively, CRISPR effector protein mRNA and guide RNA can beadministered together. Advantageously, a second booster dose of guideRNA can be administered 1-12 hours (preferably around 2-6 hours) afterthe initial administration of CRISPR effector protein mRNA +guide RNA.

The CRISPR effector protein of the present invention, i.e. a C2c2effector protein is sometimes referred to herein as a CRISPR Enzyme. Itwill be appreciated that the effector protein is based on or derivedfrom an enzyme, so the term ‘effector protein’ certainly includes‘enzyme’ in some embodiments. However, it will also be appreciated thatthe effector protein may, as required in some embodiments, have DNA orRNA binding, but not necessarily cutting or nicking, activity, includinga dead-Cas effector protein function.

Additional administrations of CRISPR effector protein mRNA and/or guideRNA might be useful to achieve the most efficient levels of genomemodification. In some embodiments, phenotypic alteration is preferablythe result of genome modification when a genetic disease is targeted,especially in methods of therapy and preferably where a repair templateis provided to correct or alter the phenotype.

In some embodiments diseases that may be targeted include thoseconcerned with disease-causing splice defects.

In some embodiments, cellular targets include HemopoieticStem/Progenitor Cells (CD34+); Human T cells; and Eye (retinalcells)—for example photoreceptor precursor cells.

In some embodiments Gene targets include: Human Beta Globin—HBB (fortreating Sickle Cell Anemia, including by stimulating gene-conversion(using closely related HBD gene as an endogenous template)); CD3(T-Cells); and CEP920—retina (eye).

In some embodiments disease targets also include: cancer; Sickle CellAnemia (based on a point mutation); HIV; Beta-Thalassemia; andophthalmic or ocular disease—for example Leber Congenital Amaurosis(LCA)-causing Splice Defect.

In some embodiments delivery methods include: Cationic Lipid Mediated“direct” delivery of Enzyme-Guide complex (RiboNucleoProtein) andelectroporation of plasmid DNA.

Inventive methods can further comprise delivery of templates, such asrepair templates, which may be dsODN or ssODN, see below. Delivery oftemplates may be via the cotemporaneous or separate from delivery of anyor all the CRISPR effector protein or guide and via the same deliverymechanism or different. In some embodiments, it is preferred that thetemplate is delivered together with the guide, and, preferably, also theCRISPR effector protein. An example may be an AAV vector.

Inventive methods can further comprise: (a) delivering to the cell adouble-stranded oligodeoxynucleotide (dsODN) comprising overhangscomplimentary to the overhangs created by said double strand break,wherein said dsODN is integrated into the locus of interest; or—(b)delivering to the cell a single-stranded oligodeoxynucleotide (ssODN),wherein said ssODN acts as a template for homology directed repair ofsaid double strand break. Inventive methods can be for the prevention ortreatment of disease in an individual, optionally wherein said diseaseis caused by a defect in said locus of interest. Inventive methods canbe conducted in vivo in the individual or ex vivo on a cell taken fromthe individual, optionally wherein said cell is returned to theindividual.

For minimization of toxicity and off-target effect, it will be importantto control the concentration of CRISPR effector protein mRNA and guideRNA delivered. Optimal concentrations of CRISPR effector protein mRNAand guide RNA can be determined by testing different concentrations in acellular or animal model and using deep sequencing the analyze theextent of modification at potential off-target genomic loci. Forexample, for the guide sequence targeting 5′-GAGTCCGAGCAGAAGAAGAA-3′ inthe EMX1 gene of the human genome, deep sequencing can be used to assessthe level of modification at the following two off-target loci, 1:5′-GAGTCCTAGCAGGAGAAGAA-3′ and 2: 5′-GAGTCTAAGCAGAAGAAGAA-3′. Theconcentration that gives the highest level of on-target modificationwhile minimizing the level of off-target modification should be chosenfor in vivo delivery.

Inducible Systems

In some embodiments, a CRISPR effector protein may form a component ofan inducible system. The inducible nature of the system would allow forspatiotemporal control of gene editing or gene expression using a formof energy. The form of energy may include but is not limited toelectromagnetic radiation, sound energy, chemical energy and thermalenergy. Examples of inducible system include tetracycline induciblepromoters (Tet-On or Tet-Off), small molecule two-hybrid transcriptionactivations systems (FKBP, ABA, etc), or light inducible systems(Phytochrome, LOV domains, or cryptochrome). In one embodiment, theCRISPR effector protein may be a part of a Light InducibleTranscriptional Effector (LITE) to direct changes in transcriptionalactivity in a sequence-specific manner. The components of a light mayinclude a CRISPR effector protein, a light-responsive cytochromeheterodimer (e.g. from Arabidopsis thaliana), and a transcriptionalactivation/repression domain. Further examples of inducible DNA bindingproteins and methods for their use are provided in U.S. 61/736,465 andU.S. 61/721,283, and WO 2014018423 A2 which is hereby incorporated byreference in its entirety.

Exemplary Methods of Using of CRISPR Cas System

The invention provides a non-naturally occurring or engineeredcomposition, or one or more polynucleotides encoding components of saidcomposition, or vector or delivery systems comprising one or morepolynucleotides encoding components of said composition for use in amodifying a target cell in vivo, ex vivo or in vitro and, may beconducted in a manner alters the cell such that once modified theprogeny or cell line of the CRISPR modified cell retains the alteredphenotype. The modified cells and progeny may be part of amulti-cellular organism such as a plant or animal with ex vivo or invivo application of CRISPR system to desired cell types. The CRISPRinvention may be a therapeutic method of treatment. The therapeuticmethod of treatment may comprise gene or genome editing, or genetherapy.

Modifying a Target with CRISPR Cas System or Complex (e.g., C2c2-RNAComplex)

In one aspect, the invention provides for methods of modifying a targetpolynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or invitro. In some embodiments, the method comprises sampling a cell orpopulation of cells from a human or non-human animal, and modifying thecell or cells. Culturing may occur at any stage ex vivo. The cell orcells may even be re-introduced into the non-human animal or plant. Forre-introduced cells it is particularly preferred that the cells are stemcells.

In some embodiments, the method comprises allowing a CRISPR complex tobind to the target polynucleotide to effect cleavage of said targetpolynucleotide thereby modifying the target polynucleotide, wherein theCRISPR complex comprises a CRISPR effector protein complexed with aguide sequence hybridized or hybridizable to a target sequence withinsaid target polynucleotide.

In one aspect, the invention provides a method of modifying expressionof a polynucleotide in a eukaryotic cell. In some embodiments, themethod comprises allowing a CRISPR complex to bind to the polynucleotidesuch that said binding results in increased or decreased expression ofsaid polynucleotide; wherein the CRISPR complex comprises a CRISPReffector protein complexed with a guide sequence hybridized orhybridizable to a target sequence within said polynucleotide. Similarconsiderations and conditions apply as above for methods of modifying atarget polynucleotide. In fact, these sampling, culturing andre-introduction options apply across the aspects of the presentinvention.

Indeed, in any aspect of the invention, the CRISPR complex may comprisea CRISPR effector protein complexed with a guide sequence hybridized orhybridizable to a target sequence. Similar considerations and conditionsapply as above for methods of modifying a target polynucleotide.

Thus in any of the non-naturally-occurring CRISPR effector proteinsdescribed herein comprise at least one modification and whereby theeffector protein has certain improved capabilities. In particular, anyof the effector proteins are capable of forming a CRISPR complex with aguide RNA. When such a complex forms, the guide RNA is capable ofbinding to a target polynucleotide sequence and the effector protein iscapable of modifying a target locus. In addition, the effector proteinin the CRISPR complex has reduced capability of modifying one or moreoff-target loci as compared to an unmodified enzyme/effector protein.

In addition, the modified CRISPR enzymes described herein encompassenzymes whereby in the CRISPR complex the effector protein has increasedcapability of modifying the one or more target loci as compared to anunmodified enzyme/effector protein. Such function may be providedseparate to or provided in combination with the above-described functionof reduced capability of modifying one or more off-target loci. Any sucheffector proteins may be provided with any of the further modificationsto the CRISPR effector protein as described herein, such as incombination with any activity provided by one or more associatedheterologous functional domains, any further mutations to reducenuclease activity and the like.

In advantageous embodiments of the invention, the modified CRISPReffector protein is provided with reduced capability of modifying one ormore off-target loci as compared to an unmodified enzyme/effectorprotein and increased capability of modifying the one or more targetloci as compared to an unmodified enzyme/effector protein. Incombination with further modifications to the effector protein,significantly enhanced specificity may be achieved. For example,combination of such advantageous embodiments with one or more additionalmutations is provided wherein the one or more additional mutations arein one or more catalytically active domains. In such effector proteins,enhanced specificity may be achieved due to an improved specificity interms of effector protein activity.

Modifications to reduce off-target effects and/or enhance on-targeteffects as described above may be made to amino acid residues located ina positively-charged region/groove situated between the RuvC-III and HNHdomains. It will be appreciated that any of the functional effectsdescribed above may be achieved by modification of amino acids withinthe aforementioned groove but also by modification of amino acidsadjacent to or outside of that groove.

Additional functionalities which may be engineered into modified CRISPReffector proteins as described herein include the following. 1. modifiedCRISPR effector proteins that disrupt DNA:protein interactions withoutaffecting protein tertiary or secondary structure. This includesresidues that contact any part of the RNA:DNA duplex. 2. modified CRISPReffector proteins that weaken intra-protein interactions holding C2c2 inconformation essential for nuclease cutting in response to DNA binding(on or off target). For example: a modification that mildly inhibits,but still allows, the nuclease conformation of the HNH domain(positioned at the scissile phosphate). 3. modified CRISPR effectorproteins that strengthen intra-protein interactions holding C2c2 in aconformation inhibiting nuclease activity in response to DNA binding (onor off targets). For example: a modification that stabilizes the HNHdomain in a conformation away from the scissile phosphate. Any suchadditional functional enhancement may be provided in combination withany other modification to the CRISPR effector protein as described indetail elsewhere herein.

Any of the herein described improved functionalities may be made to anyCRISPR effector protein, such as a C2c2 effector protein. However, itwill be appreciated that any of the functionalities described herein maybe engineered into C2c2 effector proteins from other orthologs,including chimeric effector proteins comprising fragments from multipleorthologs.

The invention uses nucleic acids to bind target DNA sequences. This isadvantageous as nucleic acids are much easier and cheaper to producethan proteins, and the specificity can be varied according to the lengthof the stretch where homology is sought. Complex 3-D positioning ofmultiple fingers, for example is not required. The terms“polynucleotide”, “nucleotide”, “nucleotide sequence”, “nucleic acid”and “oligonucleotide” are used interchangeably. They refer to apolymeric form of nucleotides of any length, either deoxyribonucleotidesor ribonucleotides, or analogs thereof. Polynucleotides may have anythree dimensional structure, and may perform any function, known orunknown. The following are non-limiting examples of polynucleotides:coding or non-coding regions of a gene or gene fragment, loci (locus)defined from linkage analysis, exons, introns, messenger RNA (mRNA),transfer RNA, ribosomal RNA, short interfering RNA (siRNA),short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. The term also encompassesnucleic-acid-like structures with synthetic backbones, see, e.g.,Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; WO 97/03211; WO96/39154; Mata, 1997; Strauss-Soukup, 1997; and Samstag, 1996. Apolynucleotide may comprise one or more modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure may be imparted before or after assembly ofthe polymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.As used herein the term “wild type” is a term of the art understood byskilled persons and means the typical form of an organism, strain, geneor characteristic as it occurs in nature as distinguished from mutant orvariant forms. A “wild type” can be a base line. As used herein the term“variant” should be taken to mean the exhibition of qualities that havea pattern that deviates from what occurs in nature. The terms“non-naturally occurring” or “engineered” are used interchangeably andindicate the involvement of the hand of man. The terms, when referringto nucleic acid molecules or polypeptides mean that the nucleic acidmolecule or the polypeptide is at least substantially free from at leastone other component with which they are naturally associated in natureand as found in nature. “Complementarity” refers to the ability of anucleic acid to form hydrogen bond(s) with another nucleic acid sequenceby either traditional Watson-Crick base pairing or other non-traditionaltypes. A percent complementarity indicates the percentage of residues ina nucleic acid molecule which can form hydrogen bonds (e.g.,Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5,6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100%complementary). “Perfectly complementary” means that all the contiguousresidues of a nucleic acid sequence will hydrogen bond with the samenumber of contiguous residues in a second nucleic acid sequence.“Substantially complementary” as used herein refers to a degree ofcomplementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or morenucleotides, or refers to two nucleic acids that hybridize understringent conditions. As used herein, “stringent conditions” forhybridization refer to conditions under which a nucleic acid havingcomplementarity to a target sequence predominantly hybridizes with thetarget sequence, and substantially does not hybridize to non-targetsequences. Stringent conditions are generally sequence-dependent, andvary depending on a number of factors. In general, the longer thesequence, the higher the temperature at which the sequence specificallyhybridizes to its target sequence. Non-limiting examples of stringentconditions are described in detail in Tijssen (1993), LaboratoryTechniques In Biochemistry And Molecular Biology-Hybridization WithNucleic Acid Probes Part I, Second Chapter “Overview of principles ofhybridization and the strategy of nucleic acid probe assay”, Elsevier,N.Y. Where reference is made to a polynucleotide sequence, thencomplementary or partially complementary sequences are also envisaged.These are preferably capable of hybridizing to the reference sequenceunder highly stringent conditions. Generally, in order to maximize thehybridization rate, relatively low-stringency hybridization conditionsare selected: about 20 to 25° C. lower than the thermal melting point(T_(m)). The T_(m) is the temperature at which 50% of specific targetsequence hybridizes to a perfectly complementary probe in solution at adefined ionic strength and pH. Generally, in order to require at leastabout 85% nucleotide complementarity of hybridized sequences, highlystringent washing conditions are selected to be about 5 to 15° C. lowerthan the T_(m). In order to require at least about 70% nucleotidecomplementarity of hybridized sequences, moderately-stringent washingconditions are selected to be about 15 to 30° C. lower than the T_(m).Highly permissive (very low stringency) washing conditions may be as lowas 50° C. below the T_(m), allowing a high level of mis-matching betweenhybridized sequences. Those skilled in the art will recognize that otherphysical and chemical parameters in the hybridization and wash stagescan also be altered to affect the outcome of a detectable hybridizationsignal from a specific level of homology between target and probesequences. Preferred highly stringent conditions comprise incubation in50% formamide, 5×SSC, and 1% SDS at 42° C., or incubation in 5×SSC and1% SDS at 65° C., with wash in 0.2×SSC and 0.1% SDS at 65° C.“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson Crick base pairing, Hoogstein binding, or inany other sequence specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combinationof these. A hybridization reaction may constitute a step in a moreextensive process, such as the initiation of PCR, or the cleavage of apolynucleotide by an enzyme. A sequence capable of hybridizing with agiven sequence is referred to as the “complement” of the given sequence.As used herein, the term “genomic locus” or “locus” (plural loci) is thespecific location of a gene or DNA sequence on a chromosome. A “gene”refers to stretches of DNA or RNA that encode a polypeptide or an RNAchain that has functional role to play in an organism and hence is themolecular unit of heredity in living organisms. For the purpose of thisinvention it may be considered that genes include regions which regulatethe production of the gene product, whether or not such regulatorysequences are adjacent to coding and/or transcribed sequences.Accordingly, a gene includes, but is not necessarily limited to,promoter sequences, terminators, translational regulatory sequences suchas ribosome binding sites and internal ribosome entry sites, enhancers,silencers, insulators, boundary elements, replication origins, matrixattachment sites and locus control regions. As used herein, “expressionof a genomic locus” or “gene expression” is the process by whichinformation from a gene is used in the synthesis of a functional geneproduct. The products of gene expression are often proteins, but innon-protein coding genes such as rRNA genes or tRNA genes, the productis functional RNA. The process of gene expression is used by all knownlife-eukaryotes (including multicellular organisms), prokaryotes(bacteria and archaea) and viruses to generate functional products tosurvive. As used herein “expression” of a gene or nucleic acidencompasses not only cellular gene expression, but also thetranscription and translation of nucleic acid(s) in cloning systems andin any other context. As used herein, “expression” also refers to theprocess by which a polynucleotide is transcribed from a DNA template(such as into and mRNA or other RNA transcript) and/or the process bywhich a transcribed mRNA is subsequently translated into peptides,polypeptides, or proteins. Transcripts and encoded polypeptides may becollectively referred to as “gene product.” If the polynucleotide isderived from genomic DNA, expression may include splicing of the mRNA ina eukaryotic cell. The terms “polypeptide”, “peptide” and “protein” areused interchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation, such asconjugation with a labeling component. As used herein the term “aminoacid” includes natural and/or unnatural or synthetic amino acids,including glycine and both the D or L optical isomers, and amino acidanalogs and peptidomimetics. As used herein, the term “domain” or“protein domain” refers to a part of a protein sequence that may existand function independently of the rest of the protein chain. Asdescribed in aspects of the invention, sequence identity is related tosequence homology. Homology comparisons may be conducted by eye, or moreusually, with the aid of readily available sequence comparison programs.These commercially available computer programs may calculate percent (%)homology between two or more sequences and may also calculate thesequence identity shared by two or more amino acid or nucleic acidsequences.

In aspects of the invention the term “guide RNA”, refers to thepolynucleotide sequence comprising one or more of a putative oridentified tracr sequence and a putative or identified crRNA sequence orguide sequence. In particular embodiments, the “guide RNA” comprises aputative or identified crRNA sequence or guide sequence. In furtherembodiments, the guide RNA does not comprise a putative or identifiedtracr sequence.

As used herein the term “wild type” is a term of the art understood byskilled persons and means the typical form of an organism, strain, geneor characteristic as it occurs in nature as distinguished from mutant orvariant forms. A “wild type” can be a base line.

As used herein the term “variant” should be taken to mean the exhibitionof qualities that have a pattern that deviates from what occurs innature.

The terms “non-naturally occurring” or “engineered” are usedinterchangeably and indicate the involvement of the hand of man. Theterms, when referring to nucleic acid molecules or polypeptides meanthat the nucleic acid molecule or the polypeptide is at leastsubstantially free from at least one other component with which they arenaturally associated in nature and as found in nature. In all aspectsand embodiments, whether they include these terms or not, it will beunderstood that, preferably, the may be optional and thus preferablyincluded or not preferably not included. Furthermore, the terms“non-naturally occurring” and “engineered” may be used interchangeablyand so can therefore be used alone or in combination and one or othermay replace mention of both together. In particular, “engineered” ispreferred in place of “non-naturally occurring” or “non-naturallyoccurring and/or engineered.”

Sequence homologies may be generated by any of a number of computerprograms known in the art, for example BLAST or FASTA, etc. A suitablecomputer program for carrying out such an alignment is the GCG WisconsinBestfit package (University of Wisconsin, U.S.A; Devereux et al., 1984,Nucleic Acids Research 12:387). Examples of other software than mayperform sequence comparisons include, but are not limited to, the BLASTpackage (see Ausubel et al., 1999 ibid—Chapter 18), FASTA (Atschul etal., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparisontools. Both BLAST and FASTA are available for offline and onlinesearching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). Howeverit is preferred to use the GCG Bestfit program. Percentage (%) sequencehomology may be calculated over contiguous sequences, i.e., one sequenceis aligned with the other sequence and each amino acid or nucleotide inone sequence is directly compared with the corresponding amino acid ornucleotide in the other sequence, one residue at a time. This is calledan “ungapped” alignment. Typically, such ungapped alignments areperformed only over a relatively short number of residues. Although thisis a very simple and consistent method, it fails to take intoconsideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion may cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without unduly penalizing the overall homology or identityscore. This is achieved by inserting “gaps” in the sequence alignment totry to maximize local homology or identity. However, these more complexmethods assign “gap penalties” to each gap that occurs in the alignmentso that, for the same number of identical amino acids, a sequencealignment with as few gaps as possible—reflecting higher relatednessbetween the two compared sequences—may achieve a higher score than onewith many gaps. “Affinity gap costs” are typically used that charge arelatively high cost for the existence of a gap and a smaller penaltyfor each subsequent residue in the gap. This is the most commonly usedgap scoring system. High gap penalties may, of course, produce optimizedalignments with fewer gaps. Most alignment programs allow the gappenalties to be modified. However, it is preferred to use the defaultvalues when using such software for sequence comparisons. For example,when using the GCG Wisconsin Bestfit package the default gap penalty foramino acid sequences is −12 for a gap and −4 for each extension.Calculation of maximum % homology therefore first requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (Devereux et al., 1984Nuc. Acids Research 12 p 387). Examples of other software than mayperform sequence comparisons include, but are not limited to, the BLASTpackage (see Ausubel et al., 1999 Short Protocols in Molecular Biology,4^(th) Ed.—Chapter 18), FASTA (Altschul et al., 1990 J. Mol. Biol.403-410) and the GENEWORKS suite of comparison tools. Both BLAST andFASTA are available for offline and online searching (see Ausubel etal., 1999, Short Protocols in Molecular Biology, pages 7-58 to 7-60).However, for some applications, it is preferred to use the GCG Bestfitprogram. A new tool, called BLAST 2 Sequences is also available forcomparing protein and nucleotide sequences (see FEMS Microbiol Lett.1999 174(2): 247-50; FEMS Microbiol Lett. 1999 177(1): 187-8 and thewebsite of the National Center for Biotechnology information at thewebsite of the National Institutes for Health). Although the final %homology may be measured in terms of identity, the alignment processitself is typically not based on an all-or-nothing pair comparison.Instead, a scaled similarity score matrix is generally used that assignsscores to each pair-wise comparison based on chemical similarity orevolutionary distance. An example of such a matrix commonly used is theBLOSUM62 matrix—the default matrix for the BLAST suite of programs. GCGWisconsin programs generally use either the public default values or acustom symbol comparison table, if supplied (see user manual for furtherdetails). For some applications, it is preferred to use the publicdefault values for the GCG package, or in the case of other software,the default matrix, such as BLOSUM62. Alternatively, percentagehomologies may be calculated using the multiple alignment feature inDNASIS™ (Hitachi Software), based on an algorithm, analogous to CLUSTAL(Higgins D G & Sharp P M (1988), Gene 73(1), 237-244). Once the softwarehas produced an optimal alignment, it is possible to calculate %homology, preferably % sequence identity. The software typically doesthis as part of the sequence comparison and generates a numericalresult. The sequences may also have deletions, insertions orsubstitutions of amino acid residues which produce a silent change andresult in a functionally equivalent substance. Deliberate amino acidsubstitutions may be made on the basis of similarity in amino acidproperties (such as polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues) and it istherefore useful to group amino acids together in functional groups.Amino acids may be grouped together based on the properties of theirside chains alone. However, it is more useful to include mutation dataas well. The sets of amino acids thus derived are likely to be conservedfor structural reasons. These sets may be described in the form of aVenn diagram (Livingstone C. D. and Barton G. J. (1993) “Proteinsequence alignments: a strategy for the hierarchical analysis of residueconservation” Comput. Appl. Biosci. 9: 745-756) (Taylor W. R. (1986)“The classification of amino acid conservation” J. Theor. Biol. 119;205-218). Conservative substitutions may be made, for example accordingto the table below which describes a generally accepted Venn diagramgrouping of amino acids.

Set Sub-set Hydrophobic F W Y H K M I L V A G C Aromatic F W Y HAliphatic I L V Polar W Y H K R E D C S T N Q Charged H K R E DPositively H K R charged Negatively E D charged Small V C A G S P T N DTiny A G S

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a vertebrate, preferably a mammal,more preferably a human. Mammals include, but are not limited to,murines, simians, humans, farm animals, sport animals, and pets.Tissues, cells and their progeny of a biological entity obtained in vivoor cultured in vitro are also encompassed.

The terms “therapeutic agent”, “therapeutic capable agent” or “treatmentagent” are used interchangeably and refer to a molecule or compound thatconfers some beneficial effect upon administration to a subject. Thebeneficial effect includes enablement of diagnostic determinations;amelioration of a disease, symptom, disorder, or pathological condition;reducing or preventing the onset of a disease, symptom, disorder orcondition; and generally counteracting a disease, symptom, disorder orpathological condition.

As used herein, “treatment” or “treating,” or “palliating” or“ameliorating” are used interchangeably. These terms refer to anapproach for obtaining beneficial or desired results including but notlimited to a therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant any therapeutically relevant improvement inor effect on one or more diseases, conditions, or symptoms undertreatment. For prophylactic benefit, the compositions may beadministered to a subject at risk of developing a particular disease,condition, or symptom, or to a subject reporting one or more of thephysiological symptoms of a disease, even though the disease, condition,or symptom may not have yet been manifested.

The term “effective amount” or “therapeutically effective amount” refersto the amount of an agent that is sufficient to effect beneficial ordesired results. The therapeutically effective amount may vary dependingupon one or more of: the subject and disease condition being treated,the weight and age of the subject, the severity of the diseasecondition, the manner of administration and the like, which can readilybe determined by one of ordinary skill in the art. The term also appliesto a dose that will provide an image for detection by any one of theimaging methods described herein. The specific dose may vary dependingon one or more of: the particular agent chosen, the dosing regimen to befollowed, whether it is administered in combination with othercompounds, timing of administration, the tissue to be imaged, and thephysical delivery system in which it is carried.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of immunology, biochemistry,chemistry, molecular biology, microbiology, cell biology, genomics andrecombinant DNA, which are within the skill of the art. See Sambrook,Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2ndedition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel,et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press,Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, ALABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).

Several aspects of the invention relate to vector systems comprising oneor more vectors, or vectors as such. Vectors can be designed forexpression of CRISPR transcripts (e.g. nucleic acid transcripts,proteins, or enzymes) in prokaryotic or eukaryotic cells. For example,CRISPR transcripts can be expressed in bacterial cells such asEscherichia coli, insect cells (using baculovirus expression vectors),yeast cells, or mammalian cells. Suitable host cells are discussedfurther in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY185, Academic Press, San Diego, Calif. (1990). Alternatively, therecombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Embodiments of the invention include sequences (both polynucleotide orpolypeptide) which may comprise homologous substitution (substitutionand replacement are both used herein to mean the interchange of anexisting amino acid residue or nucleotide, with an alternative residueor nucleotide) that may occur i.e., like-for-like substitution in thecase of amino acids such as basic for basic, acidic for acidic, polarfor polar, etc. Non-homologous substitution may also occur i.e., fromone class of residue to another or alternatively involving the inclusionof unnatural amino acids such as ornithine (hereinafter referred to asZ), diaminobutyric acid ornithine (hereinafter referred to as B),norleucine ornithine (hereinafter referred to as O), pyriylalanine,thienylalanine, naphthylalanine and phenylglycine. Variant amino acidsequences may include suitable spacer groups that may be insertedbetween any two amino acid residues of the sequence including alkylgroups such as methyl, ethyl or propyl groups in addition to amino acidspacers such as glycine or β-alanine residues. A further form ofvariation, which involves the presence of one or more amino acidresidues in peptoid form, may be well understood by those skilled in theart. For the avoidance of doubt, “the peptoid form” is used to refer tovariant amino acid residues wherein the α-carbon substituent group is onthe residue's nitrogen atom rather than the α-carbon. Processes forpreparing peptides in the peptoid form are known in the art, for exampleSimon R J et al., PNAS (1992) 89(20), 9367-9371 and Horwell D C, TrendsBiotechnol. (1995) 13(4), 132-134.

Homology modelling: Corresponding residues in other C2c2 orthologs canbe identified by the methods of Zhang et al., 2012 (Nature; 490(7421):556-60) and Chen et al., 2015 (PLoS Comput Biol; 11(5): e1004248)—acomputational protein-protein interaction (PPI) method to predictinteractions mediated by domain-motif interfaces. PrePPI (PredictingPPI), a structure based PPI prediction method, combines structuralevidence with non-structural evidence using a Bayesian statisticalframework. The method involves taking a pair a query proteins and usingstructural alignment to identify structural representatives thatcorrespond to either their experimentally determined structures orhomology models. Structural alignment is further used to identify bothclose and remote structural neighbors by considering global and localgeometric relationships. Whenever two neighbors of the structuralrepresentatives form a complex reported in the Protein Data Bank, thisdefines a template for modelling the interaction between the two queryproteins. Models of the complex are created by superimposing therepresentative structures on their corresponding structural neighbor inthe template. This approach is further described in Dey et al., 2013(Prot Sci; 22: 359-66).

For purpose of this invention, amplification means any method employinga primer and a polymerase capable of replicating a target sequence withreasonable fidelity. Amplification may be carried out by natural orrecombinant DNA polymerases such as TaqGold™, T7 DNA polymerase, Klenowfragment of E. coli DNA polymerase, and reverse transcriptase. Apreferred amplification method is PCR.

In certain aspects the invention involves vectors. A used herein, a“vector” is a tool that allows or facilitates the transfer of an entityfrom one environment to another. It is a replicon, such as a plasmid,phage, or cosmid, into which another DNA segment may be inserted so asto bring about the replication of the inserted segment. Generally, avector is capable of replication when associated with the proper controlelements. In general, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. Vectors include, but are not limited to, nucleic acidmolecules that are single-stranded, double-stranded, or partiallydouble-stranded; nucleic acid molecules that comprise one or more freeends, no free ends (e.g., circular); nucleic acid molecules thatcomprise DNA, RNA, or both; and other varieties of polynucleotides knownin the art. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments canbe inserted, such as by standard molecular cloning techniques. Anothertype of vector is a viral vector, wherein virally-derived DNA or RNAsequences are present in the vector for packaging into a virus (e.g.,retroviruses, replication defective retroviruses, adenoviruses,replication defective adenoviruses, and adeno-associated viruses(AAVs)). Viral vectors also include polynucleotides carried by a virusfor transfection into a host cell. Certain vectors are capable ofautonomous replication in a host cell into which they are introduced(e.g., bacterial vectors having a bacterial origin of replication andepisomal mammalian vectors). Other vectors (e.g., non-episomal mammalianvectors) are integrated into the genome of a host cell upon introductioninto the host cell, and thereby are replicated along with the hostgenome. Moreover, certain vectors are capable of directing theexpression of genes to which they are operatively-linked. Such vectorsare referred to herein as “expression vectors.” Common expressionvectors of utility in recombinant DNA techniques are often in the formof plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.,in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell). With regards torecombination and cloning methods, mention is made of U.S. patentapplication Ser. No. 10/815,730, published Sep. 2, 2004 as US2004-0171156 A1, the contents of which are herein incorporated byreference in their entirety.

Aspects of the invention relate to bicistronic vectors for guide RNA andwild type, modified or mutated CRISPR effector proteins/enzymes (e.g.C2c2). Bicistronic expression vectors guide RNA and wild type, modifiedor mutated CRISPR effector proteins/enzymes (e.g. C2c2) are preferred.In general and particularly in this embodiment and wild type, modifiedor mutated CRISPR effector proteins/enzymes (e.g. C2c2) is preferablydriven by the CBh promoter. The RNA may preferably be driven by a PolIII promoter, such as a U6 promoter. Ideally the two are combined.

In some embodiments, a loop in the guide RNA is provided. This may be astem loop or a tetra loop. The loop is preferably GAAA, but it is notlimited to this sequence or indeed to being only 4 bp in length. Indeed,preferred loop forming sequences for use in hairpin structures are fournucleotides in length, and most preferably have the sequence GAAA.However, longer or shorter loop sequences may be used, as mayalternative sequences. The sequences preferably include a nucleotidetriplet (for example, AAA), and an additional nucleotide (for example Cor G). Examples of loop forming sequences include CAAA and AAAG.

In practicing any of the methods disclosed herein, a suitable vector canbe introduced to a cell or an embryo via one or more methods known inthe art, including without limitation, microinjection, electroporation,sonoporation, biolistics, calcium phosphate-mediated transfection,cationic transfection, liposome transfection, dendrimer transfection,heat shock transfection, nucleofection transfection, magnetofection,lipofection, impalefection, optical transfection, proprietaryagent-enhanced uptake of nucleic acids, and delivery via liposomes,immunoliposomes, virosomes, or artificial virions. In some methods, thevector is introduced into an embryo by microinjection. The vector orvectors may be microinjected into the nucleus or the cytoplasm of theembryo. In some methods, the vector or vectors may be introduced into acell by nucleofection.

The term “regulatory element” is intended to include promoters,enhancers, internal ribosomal entry sites (IRES), and other expressioncontrol elements (e.g., transcription termination signals, such aspolyadenylation signals and poly-U sequences). Such regulatory elementsare described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).Regulatory elements include those that direct constitutive expression ofa nucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). A tissue-specific promoter maydirect expression primarily in a desired tissue of interest, such asmuscle, neuron, bone, skin, blood, specific organs (e.g., liver,pancreas), or particular cell types (e.g., lymphocytes). Regulatoryelements may also direct expression in a temporal-dependent manner, suchas in a cell-cycle dependent or developmental stage-dependent manner,which may or may not also be tissue or cell-type specific. In someembodiments, a vector comprises one or more pol III promoter (e.g., 1,2, 3, 4, 5, or more pol III promoters), one or more pol II promoters(e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol Ipromoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), orcombinations thereof. Examples of pol III promoters include, but are notlimited to, U6 and H1 promoters. Examples of pol II promoters include,but are not limited to, the retroviral Rous sarcoma virus (RSV) LTRpromoter (optionally with the RSV enhancer), the cytomegalovirus (CMV)promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al,Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductasepromoter, the f3-actin promoter, the phosphoglycerol kinase (PGK)promoter, and the EF1α promoter. Also encompassed by the term“regulatory element” are enhancer elements, such as WPRE; CMV enhancers;the R-U5′ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p.466-472, 1988); SV40 enhancer; and the intron sequence between exons 2and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p.1527-31, 1981). It will be appreciated by those skilled in the art thatthe design of the expression vector can depend on such factors as thechoice of the host cell to be transformed, the level of expressiondesired, etc. A vector can be introduced into host cells to therebyproduce transcripts, proteins, or peptides, including fusion proteins orpeptides, encoded by nucleic acids as described herein (e.g., clusteredregularly interspersed short palindromic repeats (CRISPR) transcripts,proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).With regards to regulatory sequences, mention is made of U.S. patentapplication Ser. No. 10/491,026, the contents of which are incorporatedby reference herein in their entirety. With regards to promoters,mention is made of PCT publication WO 2011/028929 and U.S. applicationSer. No. 12/511,940, the contents of which are incorporated by referenceherein in their entirety.

Vectors can be designed for expression of CRISPR transcripts (e.g.,nucleic acid transcripts, proteins, or enzymes) in prokaryotic oreukaryotic cells. For example, CRISPR transcripts can be expressed inbacterial cells such as Escherichia coli, insect cells (usingbaculovirus expression vectors), yeast cells, or mammalian cells.Suitable host cells are discussed further in Goeddel, GENE EXPRESSIONTECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.(1990). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Vectors may be introduced and propagated in a prokaryote or prokaryoticcell. In some embodiments, a prokaryote is used to amplify copies of avector to be introduced into a eukaryotic cell or as an intermediatevector in the production of a vector to be introduced into a eukaryoticcell (e.g., amplifying a plasmid as part of a viral vector packagingsystem). In some embodiments, a prokaryote is used to amplify copies ofa vector and express one or more nucleic acids, such as to provide asource of one or more proteins for delivery to a host cell or hostorganism. Expression of proteins in prokaryotes is most often carriedout in Escherichia coli with vectors containing constitutive orinducible promoters directing the expression of either fusion ornon-fusion proteins. Fusion vectors add a number of amino acids to aprotein encoded therein, such as to the amino terminus of therecombinant protein. Such fusion vectors may serve one or more purposes,such as: (i) to increase expression of recombinant protein; (ii) toincrease the solubility of the recombinant protein; and (iii) to aid inthe purification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase. Example fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,Academic Press, San Diego, Calif. (1990) 60-89).

In some embodiments, a vector is a yeast expression vector. Examples ofvectors for expression in yeast Saccharomyces cerivisae include pYepSec1(Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kuijan andHerskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), andpicZ (InVitrogen Corp, San Diego, Calif.).

In some embodiments, a vector drives protein expression in insect cellsusing baculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., SF9 cells)include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39).

In some embodiments, a vector is capable of driving expression of one ormore sequences in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, 1987.Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195).When used in mammalian cells, the expression vector's control functionsare typically provided by one or more regulatory elements. For example,commonly used promoters are derived from polyoma, adenovirus 2,cytomegalovirus, simian virus 40, and others disclosed herein and knownin the art. For other suitable expression systems for both prokaryoticand eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al.,MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989.

In some embodiments, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert, et al.,1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame andEaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) andimmunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen andBaltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci.USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985.Science 230: 912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990.Science 249: 374-379) and the α-fetoprotein promoter (Campes andTilghman, 1989. Genes Dev. 3: 537-546). With regards to theseprokaryotic and eukaryotic vectors, mention is made of U.S. Pat. No.6,750,059, the contents of which are incorporated by reference herein intheir entirety. Other embodiments of the invention may relate to the useof viral vectors, with regards to which mention is made of U.S. patentapplication Ser. No. 13/092,085, the contents of which are incorporatedby reference herein in their entirety. Tissue-specific regulatoryelements are known in the art and in this regard, mention is made ofU.S. Pat. No. 7,776,321, the contents of which are incorporated byreference herein in their entirety.

In some embodiments, a regulatory element is operably linked to one ormore elements of a CRISPR system so as to drive expression of the one ormore elements of the CRISPR system. In general, CRISPRs (ClusteredRegularly Interspaced Short Palindromic Repeats), also known as SPIDRs(SPacer Interspersed Direct Repeats), constitute a family of DNA locithat are usually specific to a particular bacterial species. The CRISPRlocus comprises a distinct class of interspersed short sequence repeats(SSRs) that were recognized in E. coli (Ishino et al., J. Bacteriol.,169:5429-5433 [1987]; and Nakata et al., J. Bacteriol., 171:3553-3556[1989]), and associated genes. Similar interspersed SSRs have beenidentified in Haloferax mediterranei, Streptococcus pyogenes, Anabaena,and Mycobacterium tuberculosis (See, Groenen et al., Mol. Microbiol.,10:1057-1065 [1993]; Hoe et al., Emerg. Infect. Dis., 5:254-263 [1999];Masepohl et al., Biochim. Biophys. Acta 1307:26-30 [1996]; and Mojica etal., Mol. Microbiol., 17:85-93 [1995]). The CRISPR loci typically differfrom other SSRs by the structure of the repeats, which have been termedshort regularly spaced repeats (SRSRs) (Janssen et al., OMICS J. Integ.Biol., 6:23-33 [2002]; and Mojica et al., Mol. Microbiol., 36:244-246[2000]). In general, the repeats are short elements that occur inclusters that are regularly spaced by unique intervening sequences witha substantially constant length (Mojica et al., [2000], supra). Althoughthe repeat sequences are highly conserved between strains, the number ofinterspersed repeats and the sequences of the spacer regions typicallydiffer from strain to strain (van Embden et al., J. Bacteriol.,182:2393-2401 [2000]). CRISPR loci have been identified in more than 40prokaryotes (See e.g., Jansen et al., Mol. Microbiol., 43:1565-1575[2002]; and Mojica et al., [2005]) including, but not limited toAeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula,Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus,Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium,Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus,Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma,Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas,Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella,Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus,Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia,Treponema, and Thermotoga.

In general, “nucleic acid-targeting system” as used in the presentapplication refers collectively to transcripts and other elementsinvolved in the expression of or directing the activity of nucleicacid-targeting CRISPR-associated (“Cas”) genes (also referred to hereinas an effector protein), including sequences encoding a nucleicacid-targeting Cas (effector) protein and a guide RNA (comprising crRNAsequence and a trans-activating CRISPR/Cas system RNA (tracrRNA)sequence), or other sequences and transcripts from a nucleicacid-targeting CRISPR locus. In some embodiments, one or more elementsof a nucleic acid-targeting system are derived from a Type V/Type VInucleic acid-targeting CRISPR system. In some embodiments, one or moreelements of a nucleic acid-targeting system is derived from a particularorganism comprising an endogenous nucleic acid-targeting CRISPR system.In general, a nucleic acid-targeting system is characterized by elementsthat promote the formation of a nucleic acid-targeting complex at thesite of a target sequence. In the context of formation of a nucleicacid-targeting complex, “target sequence” refers to a sequence to whicha guide sequence is designed to have complementarity, wherehybridization between a target sequence and a guide RNA promotes theformation of a DNA or RNA-targeting complex. Full complementarity is notnecessarily required, provided there is sufficient complementarity tocause hybridization and promote formation of a nucleic acid-targetingcomplex. A target sequence may comprise RNA polynucleotides. In someembodiments, a target sequence is located in the nucleus or cytoplasm ofa cell. In some embodiments, the target sequence may be within anorganelle of a eukaryotic cell, for example, mitochondrion orchloroplast. A sequence or template that may be used for recombinationinto the targeted locus comprising the target sequences is referred toas an “editing template” or “editing RNA” or “editing sequence”. Inaspects of the invention, an exogenous template RNA may be referred toas an editing template. In an aspect of the invention the recombinationis homologous recombination.

Typically, in the context of an endogenous nucleic acid-targetingsystem, formation of a nucleic acid-targeting complex (comprising aguide RNA hybridized to a target sequence and complexed with one or morenucleic acid-targeting effector proteins) results in cleavage of one orboth RNA strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 50, or more base pairs from) the target sequence. In someembodiments, one or more vectors driving expression of one or moreelements of a nucleic acid-targeting system are introduced into a hostcell such that expression of the elements of the nucleic acid-targetingsystem direct formation of a nucleic acid-targeting complex at one ormore target sites. For example, a nucleic acid-targeting effectorprotein and a guide RNA could each be operably linked to separateregulatory elements on separate vectors. Alternatively, two or more ofthe elements expressed from the same or different regulatory elements,may be combined in a single vector, with one or more additional vectorsproviding any components of the nucleic acid-targeting system notincluded in the first vector. nucleic acid-targeting system elementsthat are combined in a single vector may be arranged in any suitableorientation, such as one element located 5′ with respect to (“upstream”of) or 3′ with respect to (“downstream” of) a second element. The codingsequence of one element may be located on the same or opposite strand ofthe coding sequence of a second element, and oriented in the same oropposite direction. In some embodiments, a single promoter drivesexpression of a transcript encoding a nucleic acid-targeting effectorprotein and a guide RNA embedded within one or more intron sequences(e.g. each in a different intron, two or more in at least one intron, orall in a single intron). In some embodiments, the nucleic acid-targetingeffector protein and guide RNA are operably linked to and expressed fromthe same promoter.

In general, a guide sequence is any polynucleotide sequence havingsufficient complementarity with a target polynucleotide sequence tohybridize with the target sequence and direct sequence-specific bindingof a nucleic acid-targeting complex to the target sequence. In someembodiments, the degree of complementarity between a guide sequence andits corresponding target sequence, when optimally aligned using asuitable alignment algorithm, is about or more than about 50%, 60%, 75%,80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may bedetermined with the use of any suitable algorithm for aligningsequences, non-limiting example of which include the Smith-Watermanalgorithm, the Needleman-Wunsch algorithm, algorithms based on theBurrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW,Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, SanDiego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq(available at maq.sourceforge.net). In some embodiments, a guidesequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75,or more nucleotides in length. In some embodiments, a guide sequence isless than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewernucleotides in length. The ability of a guide sequence to directsequence-specific binding of a nucleic acid-targeting complex to atarget sequence may be assessed by any suitable assay. For example, thecomponents of a nucleic acid-targeting system sufficient to form anucleic acid-targeting complex, including the guide sequence to betested, may be provided to a host cell having the corresponding targetsequence, such as by transfection with vectors encoding the componentsof the nucleic acid-targeting CRISPR sequence, followed by an assessmentof preferential cleavage within or in the vicinity of the targetsequence, such as by Surveyor assay as described herein. Similarly,cleavage of a target polynucleotide sequence (or a sequence in thevicinity thereof) may be evaluated in a test tube by providing thetarget sequence, components of a nucleic acid-targeting complex,including the guide sequence to be tested and a control guide sequencedifferent from the test guide sequence, and comparing binding or rate ofcleavage at or in the vicinity of the target sequence between the testand control guide sequence reactions. Other assays are possible, andwill occur to those skilled in the art.

A guide sequence may be selected to target any target sequence. In someembodiments, the target sequence is a sequence within a gene transcriptor mRNA.

In some embodiments, the target sequence is a sequence within a genomeof a cell.

In some embodiments, a guide sequence is selected to reduce the degreeof secondary structure within the guide sequence. Secondary structuremay be determined by any suitable polynucleotide folding algorithm. Someprograms are based on calculating the minimal Gibbs free energy. Anexample of one such algorithm is mFold, as described by Zuker andStiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example foldingalgorithm is the online webserver RNAfold, developed at Institute forTheoretical Chemistry at the University of Vienna, using the centroidstructure prediction algorithm (see e.g. A. R. Gruber et al., 2008, Cell106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology27(12): 1151-62). Further algorithms may be found in U.S. applicationSer. No. TBA (attorney docket 44790.11.2022; Broad ReferenceBI-2013/004A); incorporated herein by reference.

In some embodiments, the nucleic acid-targeting effector protein is partof a fusion protein comprising one or more heterologous protein domains(e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or moredomains in addition to the nucleic acid-targeting effector protein). Insome embodiments, the CRISPR effector protein/enzyme is part of a fusionprotein comprising one or more heterologous protein domains (e.g. aboutor more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains inaddition to the CRISPR enzyme). A CRISPR effector protein/enzyme fusionprotein may comprise any additional protein sequence, and optionally alinker sequence between any two domains. Examples of protein domainsthat may be fused to an effector protein include, without limitation,epitope tags, reporter gene sequences, and protein domains having one ormore of the following activities: methylase activity, demethylaseactivity, transcription activation activity, transcription repressionactivity, transcription release factor activity, histone modificationactivity, RNA cleavage activity and nucleic acid binding activity.Non-limiting examples of epitope tags include histidine (His) tags, V5tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-Gtags, and thioredoxin (Trx) tags. Examples of reporter genes include,but are not limited to, glutathione-S-transferase (GST), horseradishperoxidase (HRP), chloramphenicol acetyltransferase (CAT)beta-galactosidase, beta-glucuronidase, luciferase, green fluorescentprotein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellowfluorescent protein (YFP), and autofluorescent proteins including bluefluorescent protein (BFP). A nucleic acid-targeting effector protein maybe fused to a gene sequence encoding a protein or a fragment of aprotein that bind DNA molecules or bind other cellular molecules,including but not limited to maltose binding protein (MBP), S-tag, Lex ADNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, andherpes simplex virus (HSV) BP16 protein fusions. Additional domains thatmay form part of a fusion protein comprising a nucleic acid-targetingeffector protein are described in US20110059502, incorporated herein byreference. In some embodiments, a tagged nucleic acid-targeting effectorprotein is used to identify the location of a target sequence.

In some embodiments, a CRISPR enzyme may form a component of aninducible system. The inducible nature of the system would allow forspatiotemporal control of gene editing or gene expression using a formof energy. The form of energy may include but is not limited toelectromagnetic radiation, sound energy, chemical energy and thermalenergy. Examples of inducible system include tetracycline induciblepromoters (Tet-On or Tet-Off), small molecule two-hybrid transcriptionactivations systems (FKBP, ABA, etc), or light inducible systems(Phytochrome, LOV domains, or cryptochrome). In one embodiment, theCRISPR enzyme may be a part of a Light Inducible TranscriptionalEffector (LITE) to direct changes in transcriptional activity in asequence-specific manner. The components of a light may include a CRISPRenzyme, a light-responsive cytochrome heterodimer (e.g. from Arabidopsisthaliana), and a transcriptional activation/repression domain. Furtherexamples of inducible DNA binding proteins and methods for their use areprovided in U.S. 61/736,465 and U.S. 61/721,283 and WO 2014/018423 andU.S. Pat. No. 8,889,418, U.S. Pat. No. 8,895,308, US20140186919,US20140242700, US20140273234, US20140335620, WO2014093635, which ishereby incorporated by reference in its entirety.

In some aspects, the invention provides methods comprising deliveringone or more polynucleotides, such as or one or more vectors as describedherein, one or more transcripts thereof, and/or one or proteinstranscribed therefrom, to a host cell. In some aspects, the inventionfurther provides cells produced by such methods, and organisms (such asanimals, plants, or fungi) comprising or produced from such cells. Insome embodiments, a nucleic acid-targeting effector protein incombination with (and optionally complexed with) a guide RNA isdelivered to a cell. Conventional viral and non-viral based genetransfer methods can be used to introduce nucleic acids in mammaliancells or target tissues. Such methods can be used to administer nucleicacids encoding components of a nucleic acid-targeting system to cells inculture, or in a host organism. Non-viral vector delivery systemsinclude DNA plasmids, RNA (e.g. a transcript of a vector describedherein), naked nucleic acid, and nucleic acid complexed with a deliveryvehicle, such as a liposome. Viral vector delivery systems include DNAand RNA viruses, which have either episomal or integrated genomes afterdelivery to the cell. For a review of gene therapy procedures, seeAnderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon,TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt,Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology andNeuroscience 8:35-36 (1995); Kremer & Perricaudet, British MedicalBulletin 51(1):31-44 (1995); Haddada et al., in Current Topics inMicrobiology and Immunology, Doerfler and Böhm (eds) (1995); and Yu etal., Gene Therapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids include lipofection,nucleofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Lipofection isdescribed in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355)and lipofection reagents are sold commercially (e.g., Transfectam™ andLipofectin™). Cationic and neutral lipids that are suitable forefficient receptor-recognition lipofection of polynucleotides includethose of Felgner, WO 91/17424; WO 91/16024. Delivery can be to cells(e.g. in vitro or ex vivo administration) or target tissues (e.g. invivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleicacids takes advantage of highly evolved processes for targeting a virusto specific cells in the body and trafficking the viral payload to thenucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro, and the modifiedcells may optionally be administered to patients (ex vivo). Conventionalviral based systems could include retroviral, lentivirus, adenoviral,adeno-associated and herpes simplex virus vectors for gene transfer.Integration in the host genome is possible with the retrovirus,lentivirus, and adeno-associated virus gene transfer methods, oftenresulting in long term expression of the inserted transgene.Additionally, high transduction efficiencies have been observed in manydifferent cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),Simian Immuno deficiency virus (SIV), human immuno deficiency virus(HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700). In applications where transient expression ispreferred, adenoviral based systems may be used. Adenoviral basedvectors are capable of very high transduction efficiency in many celltypes and do not require cell division. With such vectors, high titerand levels of expression have been obtained. This vector can be producedin large quantities in a relatively simple system. Adeno-associatedvirus (“AAV”) vectors may also be used to transduce cells with targetnucleic acids, e.g., in the in vitro production of nucleic acids andpeptides, and for in vivo and ex vivo gene therapy procedures (see,e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368;WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J.Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectorsare described in a number of publications, including U.S. Pat. No.5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985);Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat &Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol.63:03822-3828 (1989).

Models of Genetic and Epigenetic Conditions

A method of the invention may be used to create a plant, an animal orcell that may be used to model and/or study genetic or epigeneticconditions of interest, such as a through a model of mutations ofinterest or a disease model. As used herein, “disease” refers to adisease, disorder, or indication in a subject. For example, a method ofthe invention may be used to create an animal or cell that comprises amodification in one or more nucleic acid sequences associated with adisease, or a plant, animal or cell in which the expression of one ormore nucleic acid sequences associated with a disease are altered. Sucha nucleic acid sequence may encode a disease associated protein sequenceor may be a disease associated control sequence. Accordingly, it isunderstood that in embodiments of the invention, a plant, subject,patient, organism or cell can be a non-human subject, patient, organismor cell. Thus, the invention provides a plant, animal or cell, producedby the present methods, or a progeny thereof. The progeny may be a cloneof the produced plant or animal, or may result from sexual reproductionby crossing with other individuals of the same species to introgressfurther desirable traits into their offspring. The cell may be in vivoor ex vivo in the cases of multicellular organisms, particularly animalsor plants. In the instance where the cell is in cultured, a cell linemay be established if appropriate culturing conditions are met andpreferably if the cell is suitably adapted for this purpose (forinstance a stem cell). Bacterial cell lines produced by the inventionare also envisaged. Hence, cell lines are also envisaged.

In some methods, the disease model can be used to study the effects ofmutations on the animal or cell and development and/or progression ofthe disease using measures commonly used in the study of the disease.Alternatively, such a disease model is useful for studying the effect ofa pharmaceutically active compound on the disease.

In some methods, the disease model can be used to assess the efficacy ofa potential gene therapy strategy. That is, a disease-associated gene orpolynucleotide can be modified such that the disease development and/orprogression is inhibited or reduced. In particular, the method comprisesmodifying a disease-associated gene or polynucleotide such that analtered protein is produced and, as a result, the animal or cell has analtered response. Accordingly, in some methods, a genetically modifiedanimal may be compared with an animal predisposed to development of thedisease such that the effect of the gene therapy event may be assessed.

In another embodiment, this invention provides a method of developing abiologically active agent that modulates a cell signaling eventassociated with a disease gene. The method comprises contacting a testcompound with a cell comprising one or more vectors that driveexpression of one or more of a CRISPR enzyme, and a direct repeatsequence linked to a guide sequence; and detecting a change in a readoutthat is indicative of a reduction or an augmentation of a cell signalingevent associated with, e.g., a mutation in a disease gene contained inthe cell.

A cell model or animal model can be constructed in combination with themethod of the invention for screening a cellular function change. Such amodel may be used to study the effects of a genome sequence modified bythe CRISPR complex of the invention on a cellular function of interest.For example, a cellular function model may be used to study the effectof a modified genome sequence on intracellular signaling orextracellular signaling. Alternatively, a cellular function model may beused to study the effects of a modified genome sequence on sensoryperception. In some such models, one or more genome sequences associatedwith a signaling biochemical pathway in the model are modified.

Several disease models have been specifically investigated. Theseinclude de novo autism risk genes CHD8, KATNAL2, and SCN2A; and thesyndromic autism (Angelman Syndrome) gene UBE3A. These genes andresulting autism models are of course preferred, but serve to show thebroad applicability of the invention across genes and correspondingmodels.

An altered expression of one or more genome sequences associated with asignalling biochemical pathway can be determined by assaying for adifference in the mRNA levels of the corresponding genes between thetest model cell and a control cell, when they are contacted with acandidate agent. Alternatively, the differential expression of thesequences associated with a signaling biochemical pathway is determinedby detecting a difference in the level of the encoded polypeptide orgene product.

To assay for an agent-induced alteration in the level of mRNAtranscripts or corresponding polynucleotides, nucleic acid contained ina sample is first extracted according to standard methods in the art.For instance, mRNA can be isolated using various lytic enzymes orchemical solutions according to the procedures set forth in Sambrook etal. (1989), or extracted by nucleic-acid-binding resins following theaccompanying instructions provided by the manufacturers. The mRNAcontained in the extracted nucleic acid sample is then detected byamplification procedures or conventional hybridization assays (e.g.Northern blot analysis) according to methods widely known in the art orbased on the methods exemplified herein.

For purpose of this invention, amplification means any method employinga primer and a polymerase capable of replicating a target sequence withreasonable fidelity. Amplification may be carried out by natural orrecombinant DNA polymerases such as TaqGold™, T7 DNA polymerase, Klenowfragment of E. coli DNA polymerase, and reverse transcriptase. Apreferred amplification method is PCR. In particular, the isolated RNAcan be subjected to a reverse transcription assay that is coupled with aquantitative polymerase chain reaction (RT-PCR) in order to quantify theexpression level of a sequence associated with a signaling biochemicalpathway.

Detection of the gene expression level can be conducted in real time inan amplification assay. In one aspect, the amplified products can bedirectly visualized with fluorescent DNA-binding agents including butnot limited to DNA intercalators and DNA groove binders. Because theamount of the intercalators incorporated into the double-stranded DNAmolecules is typically proportional to the amount of the amplified DNAproducts, one can conveniently determine the amount of the amplifiedproducts by quantifying the fluorescence of the intercalated dye usingconventional optical systems in the art. DNA-binding dye suitable forthis application include SYBR green, SYBR blue, DAPI, propidium iodine,Hoeste, SYBR gold, ethidium bromide, acridines, proflavine, acridineorange, acriflavine, fluorcoumanin, ellipticine, daunomycin,chloroquine, distamycin D, chromomycin, homidium, mithramycin, rutheniumpolypyridyls, anthramycin, and the like.

In another aspect, other fluorescent labels such as sequence specificprobes can be employed in the amplification reaction to facilitate thedetection and quantification of the amplified products. Probe-basedquantitative amplification relies on the sequence-specific detection ofa desired amplified product. It utilizes fluorescent, target-specificprobes (e.g., TaqMan® probes) resulting in increased specificity andsensitivity. Methods for performing probe-based quantitativeamplification are well established in the art and are taught in U.S.Pat. No. 5,210,015.

In yet another aspect, conventional hybridization assays usinghybridization probes that share sequence homology with sequencesassociated with a signaling biochemical pathway can be performed.Typically, probes are allowed to form stable complexes with thesequences associated with a signaling biochemical pathway containedwithin the biological sample derived from the test subject in ahybridization reaction. It will be appreciated by one of skill in theart that where antisense is used as the probe nucleic acid, the targetpolynucleotides provided in the sample are chosen to be complementary tosequences of the antisense nucleic acids. Conversely, where thenucleotide probe is a sense nucleic acid, the target polynucleotide isselected to be complementary to sequences of the sense nucleic acid.

Hybridization can be performed under conditions of various stringency.Suitable hybridization conditions for the practice of the presentinvention are such that the recognition interaction between the probeand sequences associated with a signaling biochemical pathway is bothsufficiently specific and sufficiently stable. Conditions that increasethe stringency of a hybridization reaction are widely known andpublished in the art. See, for example, (Sambrook, et al., (1989);Nonradioactive In Situ Hybridization Application Manual, BoehringerMannheim, second edition). The hybridization assay can be formed usingprobes immobilized on any solid support, including but are not limitedto nitrocellulose, glass, silicon, and a variety of gene arrays. Apreferred hybridization assay is conducted on high-density gene chips asdescribed in U.S. Pat. No. 5,445,934.

For a convenient detection of the probe-target complexes formed duringthe hybridization assay, the nucleotide probes are conjugated to adetectable label. Detectable labels suitable for use in the presentinvention include any composition detectable by photochemical,biochemical, spectroscopic, immunochemical, electrical, optical orchemical means. A wide variety of appropriate detectable labels areknown in the art, which include fluorescent or chemiluminescent labels,radioactive isotope labels, enzymatic or other ligands. In preferredembodiments, one will likely desire to employ a fluorescent label or anenzyme tag, such as digoxigenin, β-galactosidase, urease, alkalinephosphatase or peroxidase, avidin/biotin complex.

The detection methods used to detect or quantify the hybridizationintensity will typically depend upon the label selected above. Forexample, radiolabels may be detected using photographic film or aphosphoimager. Fluorescent markers may be detected and quantified usinga photodetector to detect emitted light. Enzymatic labels are typicallydetected by providing the enzyme with a substrate and measuring thereaction product produced by the action of the enzyme on the substrate;and finally colorimetric labels are detected by simply visualizing thecolored label.

An agent-induced change in expression of sequences associated with asignaling biochemical pathway can also be determined by examining thecorresponding gene products. Determining the protein level typicallyinvolves a) contacting the protein contained in a biological sample withan agent that specifically bind to a protein associated with a signalingbiochemical pathway; and (b) identifying any agent:protein complex soformed. In one aspect of this embodiment, the agent that specificallybinds a protein associated with a signaling biochemical pathway is anantibody, preferably a monoclonal antibody.

The reaction is performed by contacting the agent with a sample of theproteins associated with a signaling biochemical pathway derived fromthe test samples under conditions that will allow a complex to formbetween the agent and the proteins associated with a signalingbiochemical pathway. The formation of the complex can be detecteddirectly or indirectly according to standard procedures in the art. Inthe direct detection method, the agents are supplied with a detectablelabel and unreacted agents may be removed from the complex; the amountof remaining label thereby indicating the amount of complex formed. Forsuch method, it is preferable to select labels that remain attached tothe agents even during stringent washing conditions. It is preferablethat the label does not interfere with the binding reaction. In thealternative, an indirect detection procedure may use an agent thatcontains a label introduced either chemically or enzymatically. Adesirable label generally does not interfere with binding or thestability of the resulting agent:polypeptide complex. However, the labelis typically designed to be accessible to an antibody for an effectivebinding and hence generating a detectable signal.

A wide variety of labels suitable for detecting protein levels are knownin the art. Non-limiting examples include radioisotopes, enzymes,colloidal metals, fluorescent compounds, bioluminescent compounds, andchemiluminescent compounds.

The amount of agent:polypeptide complexes formed during the bindingreaction can be quantified by standard quantitative assays. Asillustrated above, the formation of agent:polypeptide complex can bemeasured directly by the amount of label remained at the site ofbinding. In an alternative, the protein associated with a signalingbiochemical pathway is tested for its ability to compete with a labeledanalog for binding sites on the specific agent. In this competitiveassay, the amount of label captured is inversely proportional to theamount of protein sequences associated with a signaling biochemicalpathway present in a test sample.

A number of techniques for protein analysis based on the generalprinciples outlined above are available in the art. They include but arenot limited to radioimmunoassays, ELISA (enzyme linked immunoradiometricassays), “sandwich” immunoassays, immunoradiometric assays, in situimmunoassays (using e.g., colloidal gold, enzyme or radioisotopelabels), western blot analysis, immunoprecipitation assays,immunofluorescent assays, and SDS-PAGE.

Antibodies that specifically recognize or bind to proteins associatedwith a signaling biochemical pathway are preferable for conducting theaforementioned protein analyses. Where desired, antibodies thatrecognize a specific type of post-translational modifications (e.g.,signaling biochemical pathway inducible modifications) can be used.Post-translational modifications include but are not limited toglycosylation, lipidation, acetylation, and phosphorylation. Theseantibodies may be purchased from commercial vendors. For example,anti-phosphotyrosine antibodies that specifically recognizetyrosine-phosphorylated proteins are available from a number of vendorsincluding Invitrogen and Perkin Elmer. Antiphosphotyrosine antibodiesare particularly useful in detecting proteins that are differentiallyphosphorylated on their tyrosine residues in response to an ER stress.Such proteins include but are not limited to eukaryotic translationinitiation factor 2 alpha (eIF-2α). Alternatively, these antibodies canbe generated using conventional polyclonal or monoclonal antibodytechnologies by immunizing a host animal or an antibody-producing cellwith a target protein that exhibits the desired post-translationalmodification.

In practicing the subject method, it may be desirable to discern theexpression pattern of an protein associated with a signaling biochemicalpathway in different bodily tissue, in different cell types, and/or indifferent subcellular structures. These studies can be performed withthe use of tissue-specific, cell-specific or subcellular structurespecific antibodies capable of binding to protein markers that arepreferentially expressed in certain tissues, cell types, or subcellularstructures.

An altered expression of a gene associated with a signaling biochemicalpathway can also be determined by examining a change in activity of thegene product relative to a control cell. The assay for an agent-inducedchange in the activity of a protein associated with a signalingbiochemical pathway will dependent on the biological activity and/or thesignal transduction pathway that is under investigation. For example,where the protein is a kinase, a change in its ability to phosphorylatethe downstream substrate(s) can be determined by a variety of assaysknown in the art. Representative assays include but are not limited toimmunoblotting and immunoprecipitation with antibodies such asanti-phosphotyrosine antibodies that recognize phosphorylated proteins.In addition, kinase activity can be detected by high throughputchemiluminescent assays such as AlphaScreen™ (available from PerkinElmer) and eTag™ assay (Chan-Hui, et al. (2003) Clinical Immunology 111:162-174).

Where the protein associated with a signaling biochemical pathway ispart of a signaling cascade leading to a fluctuation of intracellular pHcondition, pH sensitive molecules such as fluorescent pH dyes can beused as the reporter molecules. In another example where the proteinassociated with a signaling biochemical pathway is an ion channel,fluctuations in membrane potential and/or intracellular ionconcentration can be monitored. A number of commercial kits andhigh-throughput devices are particularly suited for a rapid and robustscreening for modulators of ion channels. Representative instrumentsinclude FLIPR™ (Molecular Devices, Inc.) and VIPR (Aurora Biosciences).These instruments are capable of detecting reactions in over 1000 samplewells of a microplate simultaneously, and providing real-timemeasurement and functional data within a second or even a minisecond.

In practicing any of the methods disclosed herein, a suitable vector canbe introduced to a cell or an embryo via one or more methods known inthe art, including without limitation, microinjection, electroporation,sonoporation, biolistics, calcium phosphate-mediated transfection,cationic transfection, liposome transfection, dendrimer transfection,heat shock transfection, nucleofection transfection, magnetofection,lipofection, impalefection, optical transfection, proprietaryagent-enhanced uptake of nucleic acids, and delivery via liposomes,immunoliposomes, virosomes, or artificial virions. In some methods, thevector is introduced into an embryo by microinjection. The vector orvectors may be microinjected into the nucleus or the cytoplasm of theembryo. In some methods, the vector or vectors may be introduced into acell by nucleofection.

The target polynucleotide of a CRISPR complex can be any polynucleotideendogenous or exogenous to the eukaryotic cell. For example, the targetpolynucleotide can be a polynucleotide residing in the nucleus of theeukaryotic cell. The target polynucleotide can be a sequence coding agene product (e.g., a protein) or a non-coding sequence (e.g., aregulatory polynucleotide or a junk DNA).

Examples of target polynucleotides include a sequence associated with asignaling biochemical pathway, e.g., a signaling biochemicalpathway-associated gene or polynucleotide. Examples of targetpolynucleotides include a disease associated gene or polynucleotide. A“disease-associated” gene or polynucleotide refers to any gene orpolynucleotide which is yielding transcription or translation productsat an abnormal level or in an abnormal form in cells derived from adisease-affected tissues compared with tissues or cells of a non diseasecontrol. It may be a gene that becomes expressed at an abnormally highlevel; it may be a gene that becomes expressed at an abnormally lowlevel, where the altered expression correlates with the occurrenceand/or progression of the disease. A disease-associated gene also refersto a gene possessing mutation(s) or genetic variation that is directlyresponsible or is in linkage disequilibrium with a gene(s) that isresponsible for the etiology of a disease. The transcribed or translatedproducts may be known or unknown, and may be at a normal or abnormallevel.

The target polynucleotide of a CRISPR complex can be any polynucleotideendogenous or exogenous to the eukaryotic cell. For example, the targetpolynucleotide can be a polynucleotide residing in the nucleus of theeukaryotic cell. The target polynucleotide can be a sequence coding agene product (e.g., a protein) or a non-coding sequence (e.g., aregulatory polynucleotide or a junk DNA). Without wishing to be bound bytheory, it is believed that the target sequence should be associatedwith a PAM (protospacer adjacent motif); that is, a short sequencerecognized by the CRISPR complex. The precise sequence and lengthrequirements for the PAM differ depending on the CRISPR enzyme used, butPAMs are typically 2-5 base pair sequences adjacent the protospacer(that is, the target sequence) Examples of PAM sequences are given inthe examples section below, and the skilled person will be able toidentify further PAM sequences for use with a given CRISPR enzyme.

The target polynucleotide of a CRISPR complex may include a number ofdisease associated genes and polynucleotides as well as signalingbiochemical pathway-associated genes and polynucleotides as listed inU.S. provisional patent applications 61/736,527 and 61/748,427 bothentitled SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATIONfiled on Dec. 12, 2012 and Jan. 2, 2013, respectively, and PCTApplication PCT/US2013/074667, entitled DELIVERY, ENGINEERING ANDOPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCEMANIPULATION AND THERAPEUTIC APPLICATIONS, filed Dec. 12, 2013, thecontents of all of which are herein incorporated by reference in theirentirety.

Examples of target polynucleotides include a sequence associated with asignaling biochemical pathway, e.g., a signaling biochemicalpathway-associated gene or polynucleotide. Examples of targetpolynucleotides include a disease associated gene or polynucleotide. A“disease-associated” gene or polynucleotide refers to any gene orpolynucleotide which is yielding transcription or translation productsat an abnormal level or in an abnormal form in cells derived from adisease-affected tissues compared with tissues or cells of a non diseasecontrol. It may be a gene that becomes expressed at an abnormally highlevel; it may be a gene that becomes expressed at an abnormally lowlevel, where the altered expression correlates with the occurrenceand/or progression of the disease. A disease-associated gene also refersto a gene possessing mutation(s) or genetic variation that is directlyresponsible or is in linkage disequilibrium with a gene(s) that isresponsible for the etiology of a disease. The transcribed or translatedproducts may be known or unknown, and may be at a normal or abnormallevel.

Genome Wide Knock-Out Screening

The CRISPR effector protein complexes described herein can be used toperform efficient and cost effective functional genomic screens. Suchscreens can utilize CRISPR effector protein based genome wide libraries.Such screens and libraries can provide for determining the function ofgenes, cellular pathways genes are involved in, and how any alterationin gene expression can result in a particular biological process. Anadvantage of the present invention is that the CRISPR system avoidsoff-target binding and its resulting side effects. This is achievedusing systems arranged to have a high degree of sequence specificity forthe target DNA. In preferred embodiments of the invention, the CRISPReffector protein complexes are C2c2 effector protein complexes.

In embodiments of the invention, a genome wide library may comprise aplurality of C2c2 guide RNAs, as described herein, comprising guidesequences that are capable of targeting a plurality of target sequencesin a plurality of genomic loci in a population of eukaryotic cells. Thepopulation of cells may be a population of embryonic stem (ES) cells.The target sequence in the genomic locus may be a non-coding sequence.The non-coding sequence may be an intron, regulatory sequence, splicesite, 3′ UTR, 5′ UTR, or polyadenylation signal. Gene function of one ormore gene products may be altered by said targeting. The targeting mayresult in a knockout of gene function. The targeting of a gene productmay comprise more than one guide RNA. A gene product may be targeted by2, 3, 4, 5, 6, 7, 8, 9, or 10 guide RNAs, preferably 3 to 4 per gene.Off-target modifications may be minimized by exploiting the staggereddouble strand breaks generated by C2c2 effector protein complexes or byutilizing methods analogous to those used in CRISPR-Cas9 systems (See,e.g., DNA targeting specificity of RNA-guided Cas9 nucleases. Hsu, P.,Scott, D., Weinstein, J., Ran, F A., Konermann, S., Agarwala, V., Li,Y., Fine, E., Wu, X., Shalem, O., Cradick, T J., Marraffini, L A., Bao,G., & Zhang, F. Nat Biotechnol doi: 10.1038/nbt.2647 (2013)),incorporated herein by reference. The targeting may be of about 100 ormore sequences. The targeting may be of about 1000 or more sequences.The targeting may be of about 20,000 or more sequences. The targetingmay be of the entire genome. The targeting may be of a panel of targetsequences focused on a relevant or desirable pathway. The pathway may bean immune pathway. The pathway may be a cell division pathway.

One aspect of the invention comprehends a genome or transcriptome widelibrary that may comprise a plurality of C2c2 guide RNAs that maycomprise guide sequences that are capable of targeting a plurality oftarget sequences in a plurality of (genomic) loci, wherein saidtargeting results in a knockout/knockdown of gene function. This librarymay potentially comprise guide RNAs that target each and every gene inthe genome of an organism.

In some embodiments of the invention the organism or subject is aeukaryote (including mammal including human) or a non-human eukaryote ora non-human animal or a non-human mammal. In some embodiments, theorganism or subject is a non-human animal, and may be an arthropod, forexample, an insect, or may be a nematode. In some methods of theinvention the organism or subject is a plant. In some methods of theinvention the organism or subject is a mammal or a non-human mammal. Anon-human mammal may be for example a rodent (preferably a mouse or arat), an ungulate, or a primate. In some methods of the invention theorganism or subject is algae, including microalgae, or is a fungus.

The knockout/knockdown of gene function may comprise: introducing intoeach cell in the population of cells a vector system of one or morevectors comprising an engineered, non-naturally occurring C2c2 effectorprotein system comprising I. a C2c2 effector protein, and II. one ormore guide RNAs, wherein components I and II may be same or on differentvectors of the system, integrating components I and II into each cell,wherein the guide sequence targets a unique gene in each cell, whereinthe C2c2 effector protein is operably linked to a regulatory element,wherein when transcribed, the guide RNA comprising the guide sequencedirects sequence-specific binding of the C2c2 effector protein system toa target sequence corresponding to the genomic loci of the unique gene,inducing cleavage of the RNA corresponding to said genomic loci by theC2c2 effector protein, and confirming different knockdown events in aplurality of unique genes in each cell of the population of cellsthereby generating a gene knockdown cell library. The inventioncomprehends that the population of cells is a population of eukaryoticcells, and in a preferred embodiment, the population of cells is apopulation of embryonic stem (ES) cells.

The one or more vectors may be plasmid vectors. The vector may be asingle vector comprising a C2c2 effector protein, a sgRNA, andoptionally, a selection marker into target cells. Not being bound by atheory, the ability to simultaneously deliver a C2c2 effector proteinand sgRNA through a single vector enables application to any cell typeof interest, without the need to first generate cell lines that expressthe C2c2 effector protein. The regulatory element may be an induciblepromoter. The inducible promoter may be a doxycycline induciblepromoter. In some methods of the invention the expression of the guidesequence is under the control of the T7 promoter and is driven by theexpression of T7 polymerase. The confirming of different knockdownevents may be by whole transcriptome sequencing. The knockout mutationmay be achieved in 100 or more unique genes. The knockdown event may beachieved in 1000 or more unique genes. The knockdown event may beachieved in 20,000 or more unique genes. The knockdown event may beachieved in the entire genome. The knockdown of gene function may beachieved in a plurality of unique genes which function in a particularphysiological pathway or condition. The pathway or condition may be animmune pathway or condition. The pathway or condition may be a celldivision pathway or condition.

The invention also provides kits that comprise the transcriptome widelibraries mentioned herein. The kit may comprise a single containercomprising vectors or plasmids comprising the library of the invention.The kit may also comprise a panel comprising a selection of unique C2c2effector protein system guide RNAs comprising guide sequences from thelibrary of the invention, wherein the selection is indicative of aparticular physiological condition. The invention comprehends that thetargeting is of about 100 or more sequences, about 1000 or moresequences or about 20,000 or more sequences or the entire transcriptome.Furthermore, a panel of target sequences may be focused on a relevant ordesirable pathway, such as an immune pathway or cell division.

In an additional aspect of the invention, the C2c2 effector protein maycomprise one or more mutations and may be used as a generic RNA bindingprotein with or without fusion to a functional domain. The mutations maybe artificially introduced mutations or gain- or loss-of-functionmutations. The mutations have been characterized as described herein. Inone aspect of the invention, the functional domain may be atranscriptional activation domain, which may be VP64. In other aspectsof the invention, the functional domain may be a transcriptionalrepressor domain, which may be KRAB or SID4X. Other aspects of theinvention relate to the mutated C2c2 effector protein being fused todomains which include but are not limited to a transcriptionalactivator, repressor, a recombinase, a transposase, a histone remodeler,a demethylase, a DNA methyltransferase, a cryptochrome, a lightinducible/controllable domain or a chemically inducible/controllabledomain. Some methods of the invention can include inducing expression oftargeted genes. In one embodiment, inducing expression by targeting aplurality of target sequences in a plurality of genomic loci in apopulation of eukaryotic cells is by use of a functional domain.

Useful in the practice of the instant invention utilizing C2c2 3effectorprotein complexes are methods used in CRISPR-Cas9 systems and referenceis made to:

Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Shalem, O.,Sanjana, N E., Hartenian, E., Shi, X., Scott, DA., Mikkelson, T., Heckl,D., Ebert, BL., Root, D E., Doench, J G., Zhang, F. Science December 12.(2013). [Epub ahead of print]; Published in final edited form as:Science. 2014 Jan. 3; 343(6166): 84-87.

Shalem et al. involves a new way to interrogate gene function on agenome-wide scale. Their studies showed that delivery of a genome-scaleCRISPR-Cas9 knockout (GeCKO) library targeted 18,080 genes with 64,751unique guide sequences enabled both negative and positive selectionscreening in human cells. First, the authors showed use of the GeCKOlibrary to identify genes essential for cell viability in cancer andpluripotent stem cells. Next, in a melanoma model, the authors screenedfor genes whose loss is involved in resistance to vemurafenib, atherapeutic that inhibits mutant protein kinase BRAF. Their studiesshowed that the highest-ranking candidates included previously validatedgenes NF 1 and MED12 as well as novel hitsNF2, CUL3, TADA2B, and TADA1.The authors observed a high level of consistency between independentguide RNAs targeting the same gene and a high rate of hit confirmation,and thus demonstrated the promise of genome-scale screening with Cas9.

Reference is also made to US patent publication number US20140357530;and PCT Patent Publication WO2014093701, hereby incorporated herein byreference.

Functional Alteration and Screening

In another aspect, the present invention provides for a method offunctional evaluation and screening of genes. The use of the CRISPRsystem of the present invention to precisely deliver functional domains,to activate or repress genes or to alter epigenetic state by preciselyaltering the methylation site on a a specific locus of interest, can bewith one or more guide RNAs applied to a single cell or population ofcells or with a library applied to genome in a pool of cells ex vivo orin vivo comprising the administration or expression of a librarycomprising a plurality of guide RNAs (sgRNAs) and wherein the screeningfurther comprises use of a C2c2 effector protein, wherein the CRISPRcomplex comprising the C2c2 effector protein is modified to comprise aheterologous functional domain. In an aspect the invention provides amethod for screening a genome/transcriptome comprising theadministration to a host or expression in a host in vivo of a library.In an aspect the invention provides a method as herein discussed furthercomprising an activator administered to the host or expressed in thehost. In an aspect the invention provides a method as herein discussedwherein the activator is attached to a C2c2 effector protein. In anaspect the invention provides a method as herein discussed wherein theactivator is attached to the N terminus or the C terminus of the C2c2effector protein. In an aspect the invention provides a method as hereindiscussed wherein the activator is attached to a sgRNA loop. In anaspect the invention provides a method as herein discussed furthercomprising a repressor administered to the host or expressed in thehost. In an aspect the invention provides a method as herein discussed,wherein the screening comprises affecting and detecting gene activation,gene inhibition, or cleavage in the locus.

In an aspect, the invention provides efficient on-target activity andminimizes off target activity. In an aspect, the invention providesefficient on-target cleavage by C2c2 effector protein and minimizesoff-target cleavage by the C2c2 effector protein. In an aspect, theinvention provides guide specific binding of C2c2 effector protein at alocus without DNA cleavage. Accordingly, in an aspect, the inventionprovides target-specific gene regulation. In an aspect, the inventionprovides guide specific binding of C2c2 effector protein at a gene locuswithout DNA cleavage. Accordingly, in an aspect, the invention providesfor cleavage at one locus and gene regulation at a different locus usinga single C2c2 effector protein. In an aspect, the invention providesorthogonal activation and/or inhibition and/or cleavage of multipletargets using one or more C2c2 effector protein and/or enzyme.

In an aspect the invention provides a method as herein discussed,wherein the host is a eukaryotic cell. In an aspect the inventionprovides a method as herein discussed, wherein the host is a mammaliancell. In an aspect the invention provides a method as herein discussed,wherein the host is a non-human eukaryote. In an aspect the inventionprovides a method as herein discussed, wherein the non-human eukaryoteis a non-human mammal. In an aspect the invention provides a method asherein discussed, wherein the non-human mammal is a mouse. An aspect theinvention provides a method as herein discussed comprising the deliveryof the C2c2 effector protein complexes or component(s) thereof ornucleic acid molecule(s) coding therefor, wherein said nucleic acidmolecule(s) are operatively linked to regulatory sequence(s) andexpressed in vivo. In an aspect the invention provides a method asherein discussed wherein the expressing in vivo is via a lentivirus, anadenovirus, or an AAV. In an aspect the invention provides a method asherein discussed wherein the delivery is via a particle, a nanoparticle,a lipid or a cell penetrating peptide (CPP).

In an aspect the invention provides a pair of CRISPR complexescomprising C2c2 effector protein, each comprising a guide RNA (sgRNA)comprising a guide sequence capable of hybridizing to a target sequencein a genomic locus of interest in a cell, wherein at least one loop ofeach sgRNA is modified by the insertion of distinct RNA sequence(s) thatbind to one or more adaptor proteins, and wherein the adaptor protein isassociated with one or more functional domains, wherein each sgRNA ofeach C2c2 effector protein complex comprises a functional domain havinga DNA cleavage activity. In an aspect the invention provides paired C2C1or C2c3 effector protein complexes as herein-discussed, wherein the DNAcleavage activity is due to a Fok1 nuclease.

In an aspect the invention provides a method for cutting a targetsequence in a genomic locus of interest comprising delivery to a cell ofthe C2c2 effector protein complexes or component(s) thereof or nucleicacid molecule(s) coding therefor, wherein said nucleic acid molecule(s)are operatively linked to regulatory sequence(s) and expressed in vivo.In an aspect the invention provides a method as herein-discussed whereinthe delivery is via a lentivirus, an adenovirus, or an AAV. In an aspectthe invention provides a method as herein-discussed or paired C2c2effector protein complexes as herein-discussed wherein the targetsequence for a first complex of the pair is on a first strand of doublestranded DNA and the target sequence for a second complex of the pair ison a second strand of double stranded DNA. In an aspect the inventionprovides a method as herein-discussed or paired C2c2 effector proteincomplexes as herein-discussed wherein the target sequences of the firstand second complexes are in proximity to each other such that the DNA iscut in a manner that facilitates homology directed repair. In an aspecta herein method can further include introducing into the cell templateDNA. In an aspect a herein method or herein paired C2c2 effector proteincomplexes can involve wherein each C2c2 effector protein complex has aC2c2 effector enzyme that is mutated such that it has no more than about5% of the nuclease activity of the C2c2 effector enzyme that is notmutated.

In an aspect the invention provides a library, method or complex asherein-discussed wherein the sgRNA is modified to have at least onenon-coding functional loop, e.g., wherein the at least one non-codingfunctional loop is repressive; for instance, wherein the at least onenon-coding functional loop comprises Alu.

In one aspect, the invention provides a method for altering or modifyingexpression of a gene product. The said method may comprise introducinginto a cell containing and expressing a DNA molecule encoding the geneproduct an engineered, non-naturally occurring CRISPR system comprisinga C2c2 effector protein and guide RNA that targets the DNA molecule,whereby the guide RNA targets the DNA molecule encoding the gene productand the C2c2 effector protein cleaves the DNA molecule encoding the geneproduct, whereby expression of the gene product is altered; and, whereinthe C2c2 effector protein and the guide RNA do not naturally occurtogether. The invention comprehends the guide RNA comprising a guidesequence linked to a direct repeat sequence. The invention furthercomprehends the C2c2 effector protein being codon optimized forexpression in a Eukaryotic cell. In a preferred embodiment theEukaryotic cell is a mammalian cell and in a more preferred embodimentthe mammalian cell is a human cell. In a further embodiment of theinvention, the expression of the gene product is decreased.

In some embodiments, one or more functional domains are associated withthe C2c2 effector protein. In some embodiments, one or more functionaldomains are associated with an adaptor protein, for example as used withthe modified guides of Konnerman et al. (Nature 517, 583-588, 29 Jan.2015). In some embodiments, one or more functional domains areassociated with an dead sgRNA (dRNA). In some embodiments, a dRNAcomplex with active C2c2 effector protein directs gene regulation by afunctional domain at on gene locus while an sgRNA directs DNA cleavageby the active C2c2 effector protein at another locus, for example asdescribed analogously in CRISPR-Cas9 systems by Dahlman et al.,‘Orthogonal gene control with a catalytically active Cas9 nuclease’ (inpress). In some embodiments, dRNAs are selected to maximize selectivityof regulation for a gene locus of interest compared to off-targetregulation. In some embodiments, dRNAs are selected to maximize targetgene regulation and minimize target cleavage

For the purposes of the following discussion, reference to a functionaldomain could be a functional domain associated with the C2c2 effectorprotein or a functional domain associated with the adaptor protein.

In some embodiments, the one or more functional domains is an NLS(Nuclear Localization Sequence) or an NES (Nuclear Export Signal). Insome embodiments, the one or more functional domains is atranscriptional activation domain comprises VP64, p65, MyoD1, HSF1, RTA,SET7/9 and a histone acetyltransferase. Other references herein toactivation (or activator) domains in respect of those associated withthe CRISPR enzyme include any known transcriptional activation domainand specifically VP64, p65, MyoD1, HSF1, RTA, SET7/9 or a histoneacetyltransferase.

In some embodiments, the one or more functional domains is atranscriptional repressor domain. In some embodiments, thetranscriptional repressor domain is a KRAB domain. In some embodiments,the transcriptional repressor domain is a NuE domain, NcoR domain, SIDdomain or a SID4X domain.

In some embodiments, the one or more functional domains have one or moreactivities comprising translation activation activity, translationrepression activity, methylase activity, demethylase activity,transcription activation activity, transcription repression activity,transcription release factor activity, histone modification activity,RNA cleavage activity, DNA cleavage activity, DNA integration activityor nucleic acid binding activity.

Histone modifying domains are also preferred in some embodiments.Exemplary histone modifying domains are discussed below. Transposasedomains, HR (Homologous Recombination) machinery domains, recombinasedomains, and/or integrase domains are also preferred as the presentfunctional domains. In some embodiments, DNA integration activityincludes HR machinery domains, integrase domains, recombinase domainsand/or transposase domains. Histone acetyltransferases are preferred insome embodiments.

In some embodiments, the DNA cleavage activity is due to a nuclease. Insome embodiments, the nuclease comprises a Fok1 nuclease. See, “DimericCRISPR RNA-guided Fold nucleases for highly specific genome editing”,Shengdar Q. Tsai, Nicolas Wyvekens, Cyd Khayter, Jennifer A. Foden,Vishal Thapar, Deepak Reyon, Mathew J. Goodwin, Martin J. Aryee, J.Keith Joung Nature Biotechnology 32(6): 569-77 (2014), relates todimeric RNA-guided Fold Nucleases that recognize extended sequences andcan edit endogenous genes with high efficiencies in human cells.

In some embodiments, the one or more functional domains is attached tothe C2c2 effector protein so that upon binding to the sgRNA and targetthe functional domain is in a spatial orientation allowing for thefunctional domain to function in its attributed function.

In some embodiments, the one or more functional domains is attached tothe adaptor protein so that upon binding of the C2c2 effector protein tothe sgRNA and target, the functional domain is in a spatial orientationallowing for the functional domain to function in its attributedfunction.

In an aspect the invention provides a composition as herein discussedwherein the one or more functional domains is attached to the C2c2effector protein or adaptor protein via a linker, optionally a GlySerlinker, as discussed herein.

Endogenous transcriptional repression is often mediated by chromatinmodifying enzymes such as histone methyltransferases (HMTs) anddeacetylases (HDACs). Repressive histone effector domains are known andan exemplary list is provided below. In the exemplary table, preferencewas given to proteins and functional truncations of small size tofacilitate efficient viral packaging (for instance via AAV). In general,however, the domains may include HDACs, histone methyltransferases(HMTs), and histone acetyltransferase (HAT) inhibitors, as well as HDACand HMT recruiting proteins. The functional domain may be or include, insome embodiments, HDAC Effector Domains, HDAC Recruiter EffectorDomains, Histone Methyltransferase (HMT) Effector Domains, HistoneMethyltransferase (HMT) Recruiter Effector Domains, or HistoneAcetyltransferase Inhibitor Effector Domains.

HDAC Effector Domains Substrate Full Selected Final Subtype/ (ifModification size truncation size Catalytic Complex Name known) (ifknown) Organism (aa) (aa) (aa) domain HDAC I HDAC8 — — X. laevis 3251-325 325 1-272: HDAC HDAC I RPD3 — — S. cerevisiae 433 19-340 32219-331: (Vannier) HDAC HDAC MesoLo4 — — M. loti 300 1-300 300 — IV(Gregoretti) HDAC HDAC — — H. sapiens 347 1-347 347 14-326: IV 11 (Gao)HDAC HD2 HDT1 — — A. thaliana 245 1-211 211 — (Wu) SIRT I SIRT3 H3K9Ac —H. sapiens 399 143-399 257 126-382: H4K16Ac (Scher) SIRT H3K56Ac SIRT IHST2 — — C. albicans 331 1-331 331 — (Hnisz) SIRT I CobB — — E. coli 2421-242 242 — (K12) (Landry) SIRT I HST2 — — S. cerevisiae 357 8-298 291 —(Wilson) SIRT SIRT5 H4K8Ac — H. sapiens 310 37-310 274 41-309: IIIH4K16Ac (Gertz) SIRT SIRT Sir2A — — P. falciparum 273 1-273 273 19-273:III (Zhu) SIRT SIRT SIRT6 H3K9Ac — H. sapiens 355 1-289 289 35-274: IVH3K56Ac (Tennen) SIRT

Accordingly, the repressor domains of the present invention may beselected from histone methyltransferases (HMTs), histone deacetylases(HDACs), histone acetyltransferase (HAT) inhibitors, as well as HDAC andHMT recruiting proteins.

The HDAC domain may be any of those in the table above, namely: HDAC8,RPD3, MesoLo4, HDAC11, HDT1, SIRT3, HST2, CobB, HST2, SIRT5, Sir2A, orSIRT6.

In some embodiment, the functional domain may be a HDAC RecruiterEffector Domain. Preferred examples include those in the Table below,namely MeCP2, MBD2b, Sin3a, NcoR, SALL1, RCOR1. NcoR is exemplified inthe present Examples and, although preferred, it is envisaged thatothers in the class will also be useful.

Table of HDAC Recruiter Effector Domains Substrate Full Selected FinalSubtype/ (if Modification size truncation size Catalytic Complex Nameknown) (if known) Organism (aa) (aa) (aa) domain Sin3a MeCP2 — — R.norvegicus 492 207-492 286 — (Nan) Sin3a MBD2b — — H. sapiens 262 45-262218 — (Boeke) Sin3a Sin3a — — H. sapiens 1273 524-851 328 627-829:(Laherty) HDAC1 interaction NcoR NcoR — — H. sapiens 2440 420-488 69 —(Zhang) NuRD SALL1 — — M. musculus 1322 1-93 93 — (Lauberth) CoRESTRCOR1 — — H. sapiens 482 81-300 220 — (Gu, Ouyang)

In some embodiment, the functional domain may be a Methyltransferase(HMT) Effector Domain. Preferred examples include those in the Tablebelow, namely NUE, vSET, EHMT2/G9A, SUV39H1, dim-5, KYP, SUVR4, SET4,SETI, SETD8, and TgSET8. NUE is exemplified in the present Examples and,although preferred, it is envisaged that others in the class will alsobe useful.

Table of Histone Methyltransferase (HMT) Effector Domains SubstrateModification Full Selected Subtype/ (if (if size truncation Final sizeCatalytic Complex Name known) known) Organism (aa) (aa) (aa) domain SETNUE H2B, — C. trachomatis 219 1-219 219 — H3, H4 (Pennini) SET vSET —H3K27me3 P. bursaria 119 1-119 119 4-112: chlorella (Mujtaba) SET2 virusSUV39 EHMT2/ H1.4K2, H3K9me1/ M. musculus 1263 969-1263 295 1025-1233:family G9A H3K9, 2, (Tachibana) preSET, H3K27 H1K25me1 SET, postSETSUV39 SUV39H1 — H3K9me2/3 H. sapiens 412 79-412 334 172-412: (Snowden)preSET, SET, postSET Suvar3-9 dim-5 — H3K9me3 N. crassa 331 1-331 33177-331: (Rathert) preSET, SET, postSET Suvar3-9 KYP — H3K9me1/2 A.thaliana 624 335-601 267 — (SUVH (Jackson) subfamily) Suvar3-9 SUVR4H3K9me1 H3K9me2/3 A. thaliana 492 180-492 313 192-462: (SUVR(Thorstensen) preSET, subfamily) SET, postSET Suvar4- SET4 — H4K20me3 C.elegans 288 1-288 288 — 20 (Vielle) SET8 SET1 — H4K20me1 C. elegans 2421-242 242 — (Vielle) SET8 SETD8 — H4K20me1 H. sapiens 393 185-393 209256-382: (Couture) SET SET8 TgSET8 — H4K20me1/ T. gondii 1893 1590-1893304 1749-1884: 2/3 (Sautel) SET

In some embodiment, the functional domain may be a HistoneMethyltransferase (HMT) Recruiter Effector Domain. Preferred examplesinclude those in the Table below, namely Hp1a, PHF19, and NIPP1.

Table of Histone Methyltransferase (HMT) Recruiter Effector DomainsSubstrate Full Selected Subtype/ (if Modification size truncation Finalsize Catalytic Complex Name known) (if known) Organism (aa) (aa) (aa)domain — Hp1a — H3K9me3 M. musculus 191 73-191 119 121-179: (Hathaway)chromoshadow — PHF19 — H3K27me3 H. sapiens 580 (1-250) + 335 163-250:GGSG (Ballaré) PHD2 linker + (500-580) — NIPP1 — H3K27me3 H. sapiens 3511-329 329 310-329: (Jin) EED

In some embodiment, the functional domain may be HistoneAcetyltransferase Inhibitor Effector Domain. Preferred examples includeSET/TAF-1β listed in the Table below.

Table of Histone Acetyltransferase Inhibitor Effector Domains SubstrateFull Selected Final Subtype/ (if Modification size truncation sizeCatalytic Complex Name known) (if known) Organism (aa) (aa) (aa) domain— SET/TAF- — — M. musculus 289 1-289 289 — 1β (Cervoni)

It is also preferred to target endogenous (regulatory) control elements,such as involved in translation, stability, or where applicable(enhancers and silencers) in addition to a promoter or promoter-proximalelements. Thus, the invention can also be used to target endogenouscontrol elements (including enhancers and silencers) in addition totargeting of the promoter. These control elements can be locatedupstream and downstream of the transcriptional start site (TSS),starting from 200 bp from the TSS to 100 kb away. Targeting of knowncontrol elements can be used to activate or repress the gene ofinterest. In some cases, a single control element can influence thetranscription of multiple target genes. Targeting of a single controlelement could therefore be used to control the transcription of multiplegenes simultaneously.

Targeting of putative control elements on the other hand (e.g. by tilingthe region of the putative control element as well as 200 bp up to 100kB around the element) can be used as a means to verify such elements(by measuring the transcription of the gene of interest) or to detectnovel control elements (e.g. by tiling 100 kb upstream and downstream ofthe TSS of the gene of interest). In addition, targeting of putativecontrol elements can be useful in the context of understanding geneticcauses of disease. Many mutations and common SNP variants associatedwith disease phenotypes are located outside coding regions. Targeting ofsuch regions with either the activation or repression systems describedherein can be followed by readout of transcription of either a) a set ofputative targets (e.g. a set of genes located in closest proximity tothe control element) or b) whole-transcriptome readout by e.g. RNAseq ormicroarray. This would allow for the identification of likely candidategenes involved in the disease phenotype. Such candidate genes could beuseful as novel drug targets.

Histone acetyltransferase (HAT) inhibitors are mentioned herein.However, an alternative in some embodiments is for the one or morefunctional domains to comprise an acetyltransferase, preferably ahistone acetyltransferase. These are useful in the field of epigenomics,for example in methods of interrogating the epigenome. Methods ofinterrogating the epigenome may include, for example, targetingepigenomic sequences. Targeting epigenomic sequences may include theguide being directed to an epigenomic target sequence. Epigenomic targetsequence may include, in some embodiments, include a promoter, silenceror an enhancer sequence.

Use of a functional domain linked to a C2c2 effector protein asdescribed herein, preferably a dead-C2c2 effector protein, morepreferably a dead-FnC2c2 effector protein, to target epigenomicsequences can be used to activate or repress promoters, silencer orenhancers.

Examples of acetyltransferases are known but may include, in someembodiments, histone acetyltransferases. In some embodiments, thehistone acetyltransferase may comprise the catalytic core of the humanacetyltransferase p300 (Gerbasch & Reddy, Nature Biotech 6 Apr. 2015).

In some preferred embodiments, the functional domain is linked to adead-C2c2 effector protein to target and activate epigenomic sequencessuch as promoters or enhancers. One or more guides directed to suchpromoters or enhancers may also be provided to direct the binding of theCRISPR enzyme to such promoters or enhancers.

In certain embodiments, the RNA targeting effector protein of theinvention can be used to interfere with co-transcriptional modificationsof DNA/chromatin structure, RNA-directed DNA methylation, orRNA-directed silencing/activation of DNA/chromatin. RNA-directed DNAmethylation (RdDM) is an epigenetic process first discovered in plants.During RdDM, double-stranded RNAs (dsRNAs) are processed to 21-24nucleotide small interfering RNAs (siRNAs) and guide methylation ofhomologous DNA loci. Besides RNA molecules, a plethora of proteins areinvolved in the establishment of RdDM, like Argonautes, DNAmethyltransferases, chromatin remodelling complexes and theplant-specific PolIV and PolV. All these act in concert to add amethyl-group at the 5′ position of cytosines. Small RNAs can modify thechromatin structure and silence transcription by guidingArgonaute-containing complexes to complementary nascent (non-coding) RNAtrancripts. Subsequently the recruitment of chromatin-modifyingcomplexes, including histone and DNA methyltransferases, is mediated.The RNA targeting effector protein of the invention may be used totarget such small RNAs and interfere in interactions between these smallRNAs and the nascent non-coding transcripts.

The term “associated with” is used here in relation to the associationof the functional domain to the C2c2 effector protein or the adaptorprotein. It is used in respect of how one molecule ‘associates’ withrespect to another, for example between an adaptor protein and afunctional domain, or between the C2c2 effector protein and a functionaldomain. In the case of such protein-protein interactions, thisassociation may be viewed in terms of recognition in the way an antibodyrecognizes an epitope. Alternatively, one protein may be associated withanother protein via a fusion of the two, for instance one subunit beingfused to another subunit. Fusion typically occurs by addition of theamino acid sequence of one to that of the other, for instance viasplicing together of the nucleotide sequences that encode each proteinor subunit. Alternatively, this may essentially be viewed as bindingbetween two molecules or direct linkage, such as a fusion protein. Inany event, the fusion protein may include a linker between the twosubunits of interest (i.e. between the enzyme and the functional domainor between the adaptor protein and the functional domain). Thus, in someembodiments, the C2c2 effector protein or adaptor protein is associatedwith a functional domain by binding thereto. In other embodiments, theC2c2 effector protein or adaptor protein is associated with a functionaldomain because the two are fused together, optionally via anintermediate linker.

Saturating Mutagenesis

The C2c2 effector protein system(s) described herein can be used toperform saturating or deep scanning mutagenesis of genomic loci inconjunction with a cellular phenotype—for instance, for determiningcritical minimal features and discrete vulnerabilities of functionalelements required for gene expression, drug resistance, and reversal ofdisease. By saturating or deep scanning mutagenesis is meant that everyor essentially every DNA base is cut within the genomic loci. A libraryof C2c2 effector protein guide RNAs may be introduced into a populationof cells. The library may be introduced, such that each cell receives asingle guide RNA (sgRNA). In the case where the library is introduced bytransduction of a viral vector, as described herein, a low multiplicityof infection (MOI) is used. The library may include sgRNAs targetingevery sequence upstream of a (protospacer adjacent motif) (PAM) sequencein a genomic locus. The library may include at least 100 non-overlappinggenomic sequences upstream of a PAM sequence for every 1000 base pairswithin the genomic locus. The library may include sgRNAs targetingsequences upstream of at least one different PAM sequence. The C2c2effector protein systems may include more than one C2c2 protein. AnyC2c2 effector protein as described herein, including orthologues orengineered C2c2 effector proteins that recognize different PAM sequencesmay be used. The frequency of off target sites for a sgRNA may be lessthan 500. Off target scores may be generated to select sgRNAs with thelowest off target sites. Any phenotype determined to be associated withcutting at a sgRNA target site may be confirmed by using sgRNAstargeting the same site in a single experiment. Validation of a targetsite may also be performed by using a modified C2c2 effector protein, asdescribed herein, and two sgRNAs targeting the genomic site of interest.Not being bound by a theory, a target site is a true hit if the changein phenotype is observed in validation experiments.

With respect to the DNA-targeting proteins disclosed herein, the genomicloci may include at least one continuous genomic region. The at leastone continuous genomic region may comprise up to the entire genome. Theat least one continuous genomic region may comprise a functional elementof the genome. The functional element may be within a non-coding region,coding gene, intronic region, promoter, or enhancer. The at least onecontinuous genomic region may comprise at least 1 kb, preferably atleast 50 kb of genomic DNA. The at least one continuous genomic regionmay comprise a transcription factor binding site. The at least onecontinuous genomic region may comprise a region of DNase Ihypersensitivity. The at least one continuous genomic region maycomprise a transcription enhancer or repressor element. The at least onecontinuous genomic region may comprise a site enriched for an epigeneticsignature. The at least one continuous genomic DNA region may comprisean epigenetic insulator. The at least one continuous genomic region maycomprise two or more continuous genomic regions that physicallyinteract. Genomic regions that interact may be determined by ‘4Ctechnology’. 4C technology allows the screening of the entire genome inan unbiased manner for DNA segments that physically interact with a DNAfragment of choice, as is described in Zhao et al. ((2006) Nat Genet 38,1341-7) and in U.S. Pat. No. 8,642,295, both incorporated herein byreference in its entirety. The epigenetic signature may be histoneacetylation, histone methylation, histone ubiquitination, histonephosphorylation, DNA methylation, or a lack thereof.

The C2c2 effector protein system(s) for saturating or deep scanningmutagenesis can be used in a population of cells. The C2c2 effectorprotein system(s) can be used in eukaryotic cells, including but notlimited to mammalian and plant cells. The population of cells may beprokaryotic cells. The population of eukaryotic cells may be apopulation of embryonic stem (ES) cells, neuronal cells, epithelialcells, immune cells, endocrine cells, muscle cells, erythrocytes,lymphocytes, plant cells, or yeast cells.

In one aspect, the present invention provides for a method of screeningfor functional elements associated with a change in a phenotype. Thelibrary may be introduced into a population of cells that are adapted tocontain a C2c2 effector protein. The cells may be sorted into at leasttwo groups based on the phenotype. The phenotype may be expression of agene, cell growth, or cell viability. The relative representation of theguide RNAs present in each group are determined, whereby genomic sitesassociated with the change in phenotype are determined by therepresentation of guide RNAs present in each group. The change inphenotype may be a change in expression of a gene of interest. The geneof interest may be upregulated, downregulated, or knocked out. The cellsmay be sorted into a high expression group and a low expression group.The population of cells may include a reporter construct that is used todetermine the phenotype. The reporter construct may include a detectablemarker. Cells may be sorted by use of the detectable marker.

In another aspect, the present invention provides for a method ofscreening for genomic sites associated with resistance to a chemicalcompound. The chemical compound may be a drug or pesticide. The librarymay be introduced into a population of cells that are adapted to containa C2c2 effector protein, wherein each cell of the population contains nomore than one guide RNA; the population of cells are treated with thechemical compound; and the representation of guide RNAs are determinedafter treatment with the chemical compound at a later time point ascompared to an early time point, whereby genomic sites associated withresistance to the chemical compound are determined by enrichment ofguide RNAs. Representation of sgRNAs may be determined by deepsequencing methods.

Useful in the practice of the instant invention utilizing C2c2effectorprotein complexes are methods used in CRISPR-Cas9 systems and referenceis made to the article entitled BCL11A enhancer dissection byCas9-mediated in situ saturating mutagenesis. Canver, M. C., Smith, E.C., Sher, F., Pinello, L., Sanjana, N. E., Shalem, O., Chen, D. D.,Schupp, P. G., Vinjamur, D. S., Garcia, S. P., Luc, S., Kurita, R.,Nakamura, Y., Fujiwara, Y., Maeda, T., Yuan, G., Zhang, F., Orkin, S.H., & Bauer, D. E. DOI:10.1038/nature15521, published online Sep. 16,2015, the article is herein incorporated by reference and discussedbriefly below:

Canver et al. involves novel pooled CRISPR-Cas9 guide RNA libraries toperform in situ saturating mutagenesis of the human and mouse BCL11Aerythroid enhancers previously identified as an enhancer associated withfetal hemoglobin (HbF) level and whose mouse ortholog is necessary forerythroid BCL11A expression. This approach revealed critical minimalfeatures and discrete vulnerabilities of these enhancers. Throughediting of primary human progenitors and mouse transgenesis, the authorsvalidated the BCL11A erythroid enhancer as a target for HbF reinduction.The authors generated a detailed enhancer map that informs therapeuticgenome editing.

Method of Using C2c2 Systems to Modify a Cell or Organism

The invention in some embodiments comprehends a method of modifying acell or organism. The cell may be a prokaryotic cell or a eukaryoticcell. The cell may be a mammalian cell. The mammalian cell many be anon-human primate, bovine, porcine, rodent or mouse cell. The cell maybe a non-mammalian eukaryotic cell such as poultry, fish or shrimp. Thecell may also be a plant cell. The plant cell may be of a crop plantsuch as cassava, corn, sorghum, wheat, or rice. The plant cell may alsobe of an algae, tree or vegetable. The modification introduced to thecell by the present invention may be such that the cell and progeny ofthe cell are altered for improved production of biologic products suchas an antibody, starch, alcohol or other desired cellular output. Themodification introduced to the cell by the present invention may be suchthat the cell and progeny of the cell include an alteration that changesthe biologic product produced.

The system may comprise one or more different vectors. In an aspect ofthe invention, the effector protein is codon optimized for expressionthe desired cell type, preferentially a eukaryotic cell, preferably amammalian cell or a human cell.

Packaging cells are typically used to form virus particles that arecapable of infecting a host cell. Such cells include 293 cells, whichpackage adenovirus, and ψ2 cells or PA317 cells, which packageretrovirus. Viral vectors used in gene therapy are usually generated byproducing a cell line that packages a nucleic acid vector into a viralparticle. The vectors typically contain the minimal viral sequencesrequired for packaging and subsequent integration into a host, otherviral sequences being replaced by an expression cassette for thepolynucleotide(s) to be expressed. The missing viral functions aretypically supplied in trans by the packaging cell line. For example, AAVvectors used in gene therapy typically only possess ITR sequences fromthe AAV genome which are required for packaging and integration into thehost genome. Viral DNA is packaged in a cell line, which contains ahelper plasmid encoding the other AAV genes, namely rep and cap, butlacking ITR sequences. The cell line may also be infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV. Additionalmethods for the delivery of nucleic acids to cells are known to thoseskilled in the art. See, for example, US20030087817, incorporated hereinby reference.

In some embodiments, a host cell is transiently or non-transientlytransfected with one or more vectors described herein. In someembodiments, a cell is transfected as it naturally occurs in a subject.In some embodiments, a cell that is transfected is taken from a subject.In some embodiments, the cell is derived from cells taken from asubject, such as a cell line. A wide variety of cell lines for tissueculture are known in the art. Examples of cell lines include, but arenot limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1,Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1,CTLL-2, CIR, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480,SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55,Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E,MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss,3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T,3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549,ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3,C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T,CHO Dhfr −/−, COR-L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7,COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3,EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa,Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812,KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231,MDA-MB-468, MDA-MB-435, MDCK II, MDCK II, MOR/0.2R, MONO-MAC 6, MTD-1A,MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3,NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F,RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line,U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, andtransgenic varieties thereof. Cell lines are available from a variety ofsources known to those with skill in the art (see, e.g., the AmericanType Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, acell transfected with one or more vectors described herein is used toestablish a new cell line comprising one or more vector-derivedsequences. In some embodiments, a cell transiently transfected with thecomponents of a nucleic acid-targeting system as described herein (suchas by transient transfection of one or more vectors, or transfectionwith RNA), and modified through the activity of a nucleic acid-targetingcomplex, is used to establish a new cell line comprising cellscontaining the modification but lacking any other exogenous sequence. Insome embodiments, cells transiently or non-transiently transfected withone or more vectors described herein, or cell lines derived from suchcells are used in assessing one or more test compounds.

In some embodiments, one or more vectors described herein are used toproduce a non-human transgenic animal or transgenic plant. In someembodiments, the transgenic animal is a mammal, such as a mouse, rat, orrabbit. In certain embodiments, the organism or subject is a plant. Incertain embodiments, the organism or subject or plant is algae. Methodsfor producing transgenic plants and animals are known in the art, andgenerally begin with a method of cell transfection, such as describedherein.

In one aspect, the invention provides for methods of modifying a targetpolynucleotide in a eukaryotic cell. In some embodiments, the methodcomprises allowing a nucleic acid-targeting complex to bind to thetarget polynucleotide to effect cleavage of said target polynucleotidethereby modifying the target polynucleotide, wherein the nucleicacid-targeting complex comprises a nucleic acid-targeting effectorprotein complexed with a guide RNA hybridized to a target sequencewithin said target polynucleotide.

In one aspect, the invention provides a method of modifying expressionof a polynucleotide in a eukaryotic cell. In some embodiments, themethod comprises allowing a nucleic acid-targeting complex to bind tothe polynucleotide such that said binding results in increased ordecreased expression of said polynucleotide; wherein the nucleicacid-targeting complex comprises a nucleic acid-targeting effectorprotein complexed with a guide RNA hybridized to a target sequencewithin said polynucleotide.

C2c2 Effector Protein Complexes can be Used in Plants

The C2c2 effector protein system(s) (e.g., single or multiplexed) can beused in conjunction with recent advances in crop genomics. The systemsdescribed herein can be used to perform efficient and cost effectiveplant gene or genome interrogation or editing or manipulation—forinstance, for rapid investigation and/or selection and/or interrogationsand/or comparison and/or manipulations and/or transformation of plantgenes or genomes; e.g., to create, identify, develop, optimize, orconfer trait(s) or characteristic(s) to plant(s) or to transform a plantgenome. There can accordingly be improved production of plants, newplants with new combinations of traits or characteristics or new plantswith enhanced traits. The C2c2 effector protein system(s) can be usedwith regard to plants in Site-Directed Integration (SDI) or Gene Editing(GE) or any Near Reverse Breeding (NRB) or Reverse Breeding (RB)techniques. Aspects of utilizing the herein described C2c2 effectorprotein systems may be analogous to the use of the CRISPR-Cas (e.g.CRISPR-Cas9) system in plants, and mention is made of the University ofArizona website “CRISPR-PLANT” (http://www.genome.arizona.edu/crispr/)(supported by Penn State and AGI). Embodiments of the invention can beused in genome editing in plants or where RNAi or similar genome editingtechniques have been used previously; see, e.g., Nekrasov, “Plant genomeediting made easy: targeted mutagenesis in model and crop plants usingthe CRISPR-Cas system,” Plant Methods 2013, 9:39(doi:10.1186/1746-4811-9-39); Brooks, “Efficient gene editing in tomatoin the first generation using the CRISPR-Cas9 system,” Plant PhysiologySeptember 2014 pp 114.247577; Shan, “Targeted genome modification ofcrop plants using a CRISPR-Cas system,” Nature Biotechnology 31, 686-688(2013); Feng, “Efficient genome editing in plants using a CRISPR/Cassystem,” Cell Research (2013) 23:1229-1232. doi:10.1038/cr.2013.114;published online 20 Aug. 2013; Xie, “RNA-guided genome editing in plantsusing a CRISPR-Cas system,” Mol Plant. 2013 November; 6(6):1975-83. doi:10.1093/mp/sst119. Epub 2013 Aug. 17; Xu, “Gene targeting using theAgrobacterium tumefaciens-mediated CRISPR-Cas system in rice,” Rice2014, 7:5 (2014), Zhou et al., “Exploiting SNPs for biallelic CRISPRmutations in the outcrossing woody perennial Populus reveals4-coumarate: CoA ligase specificity and Redundancy,” New Phytologist(2015) (Forum) 1-4 (available online only at www.newphytologist.com);Caliando et al, “Targeted DNA degradation using a CRISPR device stablycarried in the host genome, NATURE COMMUNICATIONS 6:6989, DOI:10.1038/ncomms7989, www.nature.com/naturecommunications DOI:10.1038/ncomms7989; U.S. Pat. No. 6,603,061-Agrobacterium-Mediated PlantTransformation Method; U.S. Pat. No. 7,868,149—Plant Genome Sequencesand Uses Thereof and US 2009/0100536—Transgenic Plants with EnhancedAgronomic Traits, all the contents and disclosure of each of which areherein incorporated by reference in their entirety. In the practice ofthe invention, the contents and disclosure of Morrell et al “Cropgenomics: advances and applications,” Nat Rev Genet. 2011 Dec. 29;13(2):85-96; each of which is incorporated by reference herein includingas to how herein embodiments may be used as to plants. Accordingly,reference herein to animal cells may also apply, mutatis mutandis, toplant cells unless otherwise apparent; and, the enzymes herein havingreduced off-target effects and systems employing such enzymes can beused in plant applciations, including those mentioned herein.

Sugano et al. (Plant Cell Physiol. 2014 March; 55(3):475-81. doi:10.1093/pcp/pcu014. Epub 2014 Jan. 18) reports the application ofCRISPR-Cas9 to targeted mutagenesis in the liverwort Marchantiapolymorpha L., which has emerged as a model species for studying landplant evolution. The U6 promoter of M. polymorpha was identified andcloned to express the gRNA. The target sequence of the gRNA was designedto disrupt the gene encoding auxin response factor 1 (ARF1) in M.polymorpha. Using Agrobacterium-mediated transformation, Sugano et al.isolated stable mutants in the gametophyte generation of M. polymorpha.CRISPR-Cas9-based site-directed mutagenesis in vivo was achieved usingeither the Cauliflower mosaic virus 35S or M. polymorpha EF1α promoterto express Cas9. Isolated mutant individuals showing an auxin-resistantphenotype were not chimeric. Moreover, stable mutants were produced byasexual reproduction of T1 plants. Multiple arf1 alleles were easilyestablished using CRIPSR/Cas9-based targeted mutagenesis. The C2c2systems of the present invention can be used to regulate the same aswell as other genes, and like expression control systesm such as RNAiand siRNA, the method of the invention can be inducible and reversible.

Kabadi et al. (Nucleic Acids Res. 2014 Oct. 29; 42(19):e147. doi: 10.1093/nar/gku749. Epub 2014 Aug. 13) developed a single lentiviral systemto express a Cas9 variant, a reporter gene and up to four sgRNAs fromindependent RNA polymerase III promoters that are incorporated into thevector by a convenient Golden Gate cloning method. Each sgRNA wasefficiently expressed and can mediate multiplex gene editing andsustained transcriptional activation in immortalized and primary humancells. The instant invention can be used to regulate the plant genes ofKabadi.

Xing et al. (BMC Plant Biology 2014, 14:327) developed a CRISPR-Cas9binary vector set based on the pGreen or pCAMBIA backbone, as well as agRNA. This toolkit requires no restriction enzymes besides BsaI togenerate final constructs harboring maize-codon optimized Cas9 and oneor more gRNAs with high efficiency in as little as one cloning step. Thetoolkit was validated using maize protoplasts, transgenic maize lines,and transgenic Arabidopsis lines and was shown to exhibit highefficiency and specificity. More importantly, using this toolkit,targeted mutations of three Arabidopsis genes were detected intransgenic seedlings of the T1 generation. Moreover, the multiple-genemutations could be inherited by the next generation. (guide RNA) modulevector set, as a toolkit for multiplex genome editing in plants. TheC2c2 systems and proteins of the instant invention may be used to targetthe genes targeted by Xing.

The C2c2 CRISPR systems of the invention may be used in the detection ofplant viruses. Gambino et al. (Phytopathology. 2006 November;96(11):1223-9. doi: 10.1094/PHYTO-96-1223) relied on amplification andmultiplex PCR for simultaneous detection of nine grapevine viruses. TheC2c2 systems and proteins of the instant invention may similarly be usedto detect multiple targets in a host. Moreover, the systems of theinvention can be used to simultaneously knock down viral gene expressionin valuable cultivars, and prevent activation or further infection bytargeting expressed vial RNA.

Murray et al. (Proc Biol Sci. 2013 Jun. 26; 280(1765):20130965. doi:10.1098/rspb.2013.0965; published 2013 Aug. 22) analyzxed 12 plant RNAviruses to investigatge evoluationary rates and found evidence ofepisodic selection possibly due to shifts between different hostgenotyopes or species. The C2c2 systems and proteins of the instantinvention may be used to tarteg or immunize against such viruses in ahost. For example, the systems of the invention can be used to blockviral RNA expression hence replication. Also, the invention can be usedto target nuclic acids for cleavage as wll as to target expression oractivation. Moreover, the systems of the invention can be multiplexed soas to hit multiple targets or multiple isolate of the same virus.

Ma et al. (Mol Plant. 2015 Aug. 3; 8(8):1274-84. doi:10.1016/j.molp.2015.04.007) reports robust CRISPR-Cas9 vector system,utilizing a plant codon optimized Cas9 gene, for convenient andhigh-efficiency multiplex genome editing in monocot and dicot plants. Maet al. designed PCR-based procedures to rapidly generate multiple sgRNAexpression cassettes, which can be assembled into the binary CRISPR-Cas9vectors in one round of cloning by Golden Gate ligation or GibsonAssembly. With this system, Ma et al. edited 46 target sites in ricewith an average 85.4% rate of mutation, mostly in biallelic andhomozygous status. Ma et al. provide examples of loss-of-function genemutations in T0 rice and T1 Arabidopsis plants by simultaneous targetingof multiple (up to eight) members of a gene family, multiple genes in abiosynthetic pathway, or multiple sites in a single gene. Similarly, theC2c2 systems of the instant invention can dffieicnelty target expressionof multiple genes simultaneously.

Lowder et al. (Plant Physiol. 2015 Aug. 21. pii: pp. 00636.2015) alsodeveloped a CRISPR-Cas9 toolbox enables multiplex genome editing andtranscriptional regulation of expressed, silenced or non-coding genes inplants. This toolbox provides researchers with a protocol and reagentsto quickly and efficiently assemble functional CRISPR-Cas9 T-DNAconstructs for monocots and dicots using Golden Gate and Gateway cloningmethods. It comes with a full suite of capabilities, includingmultiplexed gene editing and transcriptional activation or repression ofplant endogenous genes. T-DNA based transformation technology isfundamental to modern plant biotechnology, genetics, molecular biologyand physiology. As such, we developed a method for the assembly of Cas9(WT, nickase or dCas9) and gRNA(s) into a T-DNA destination-vector ofinterest. The assembly method is based on both Golden Gate assembly andMultiSite Gateway recombination. Three modules are required forassembly. The first module is a Cas9 entry vector, which containspromoterless Cas9 or its derivative genes flanked by attL1 and attR5sites. The second module is a gRNA entry vector which contains entrygRNA expression cassettes flanked by attL5 and attL2 sites. The thirdmodule includes attR1-attR2-containing destination T-DNA vectors thatprovide promoters of choice for Cas9 expression. The toolbox of Lowderet al. may be applied to the C2c2 effector protein system of the presentinvention.

Organisms such as yeast and microalgae are widely used for syntheticbiology. Stovicek et al. (Metab. Eng. Comm., 2015; 2:13 describes genomeediting of industrial yeast, for example, Saccharomyces cerevisae, toefficiently produce robust strains for industrial production. Stovicekused a CRISPR-Cas9 system codon-optimized for yeast to simultaneouslydisrupt both alleles of an endogenous gene and knock in a heterologousgene. Cas9 and gRNA were expressed from genomic or episomal 2μ-basedvector locations. The authors also showed that gene disruptionefficiency could be improved by optimization of the levels of Cas9 andgRNA expression. Hlavová et al. (Biotechnol. Adv. 2015) discussesdevelopment of species or strains of microalgae using techniques such asCRISPR to target nuclear and chloroplast genes for insertionalmutagenesis and screening. The same plasmids and vectors can be applicedto the C2c2 systems of the instant invention.

Petersen (“Towards precisely glycol engineered plants,” Plant BiotechDenmark Annual meeting 2015, Copenhagen, Denmark) developed a method ofusing CRISPR/Cas9 to engineer genome changes in Arabidopsis, for exampleto glyco engineer Arabidopsis for production of proteins and productshaving desired posttranslational modifications. Hebelstrup et al. (FrontPlant Sci. 2015 Apr. 23; 6:247) outlines in planta starchbioengineering, providing crops that express starch modifying enzymesand directly produce products that normally are made by industrialchemical and/or physical treatments of starches. The methods of Petersenand Hebelstrup may be applied to the C2c2 effector protein system of thepresent invention.

Kurthe t al, J Virol. 2012 June; 86(11):6002-9. doi:10.1128/JVI.00436-12. Epub 2012 Mar. 21) developed an RNA virus-basedvector for the introduction of desired traits into grapevine withoutheritable modifications to the genome. The vector provided the abilityto regulate expression of of endogenous genes by virus-induced genesilencing. The C2c2 systems and proteins of the instant invention can beused to silence genes and proteins without heritable modification to thegenome.

In an embodiment, the plant may be a legume. The present invention mayutilize the herein disclosed CRISP-Cas system for exploring andmodifying, for example, without limitation, soybeans, peas, and peanuts.Curtin et al. provides a toolbox for legume function genomics. (SeeCurtin et al., “A genome engineering toolbox for legume Functionalgenomics,” International Plant and Animal Genome Conference XXII 2014).Curtin used the genetic transformation of CRISPR to knock-out/downsingle copy and duplicated legume genes both in hairy root and wholeplant systems. Some of the target genes were chosen in order to exploreand optimize the features of knock-out/down systems (e.g., phytoenedesaturase), while others were identified by soybean homology toArabidopsis Dicer-like genes or by genome-wide association studies ofnodulation in Medicago. The C2c2 systems and proteins of the instantinvention can be used to knockout/knockdown systems.

Peanut allergies and allergies to legumes generally are a real andserious health concern. The C2c2 effector protein system of the presentinvention can be used to identify and then edit or silence genesencoding allergenic proteins of such legumes. Without limitation as tosuch genes and proteins, Nicolaou et al. identifies allergenic proteinsin peanuts, soybeans, lentils, peas, lupin, green beans, and mung beans.See, Nicolaou et al., Current Opinion in Allergy and Clinical Immunology2011; 11(3):222).

In an advantageous embodiment, the plant may be a tree. The presentinvention may also utilize the herein disclosed CRISPR Cas system forherbaceous systems (see, e.g., Belhaj et al., Plant Methods 9: 39 andHarrison et al., Genes & Development 28: 1859-1872). In a particularlyadvantageous embodiment, the CRISPR Cas system of the present inventionmay target single nucleotide polymorphisms (SNPs) in trees (see, e.g.,Zhou et al., New Phytologist, Volume 208, Issue 2, pages 298-301,October 2015). In the Zhou et al. study, the authors applied a CRISPRCas system in the woody perennial Populus using the 4-coumarate:CoAligase (4CL) gene family as a case study and achieved 100% mutationalefficiency for two 4CL genes targeted, with every transformant examinedcarrying biallelic modifications. In the Zhou et al., study, theCRISPR-Cas9 system was highly sensitive to single nucleotidepolymorphisms (SNPs), as cleavage for a third 4CL gene was abolished dueto SNPs in the target sequence. These methods may be applied to the C2c2effector protein system of the present invention.

The methods of Zhou et al. (New Phytologist, Volume 208, Issue 2, pages298-301, October 2015) may be applied to the present invention asfollows. Two 4CL genes, 4CL1 and 4CL2, associated with lignin andflavonoid biosynthesis, respectively are targeted for CRISPR-Cas9editing. The Populus tremula×alba clone 717-1B4 routinely used fortransformation is divergent from the genome-sequenced Populustrichocarpa. Therefore, the 4CL1 and 4CL2 gRNAs designed from thereference genome are interrogated with in-house 717 RNA-Seq data toensure the absence of SNPs which could limit Cas efficiency. A thirdgRNA designed for 4CL5, a genome duplicate of 4CL1, is also included.The corresponding 717 sequence harbors one SNP in each allelenear/within the PAM, both of which are expected to abolish targeting bythe 4CL5-gRNA. All three gRNA target sites are located within the firstexon. For 717 transformation, the gRNA is expressed from the MedicagoU6.6 promoter, along with a human codon-optimized Cas under control ofthe CaMV 35S promoter in a binary vector. Transformation with theCas-only vector can serve as a control. Randomly selected 4CL1 and 4CL2lines are subjected to amplicon-sequencing. The data is then processedand biallelic mutations are confirmed in all cases. These methods may beapplied to the C2c2 effector protein system of the present invention.

In plants, pathogens are often host-specific. For example, Fusariumoxysporum f. sp. lycopersici causes tomato wilt but attacks only tomato,and F. oxysporum f. dianthii Puccinia graminis f. sp. tritici attacksonly wheat. Plants have existing and induced defenses to resist mostpathogens. Mutations and recombination events across plant generationslead to genetic variability that gives rise to susceptibility,especially as pathogens reproduce with more frequency than plants. Inplants there can be non-host resistance, e.g., the host and pathogen areincompatible. There can also be Horizontal Resistance, e.g., partialresistance against all races of a pathogen, typically controlled by manygenes and Vertical Resistance, e.g., complete resistance to some racesof a pathogen but not to other races, typically controlled by a fewgenes. In a Gene-for-Gene level, plants and pathogens evolve together,and the genetic changes in one balance changes in other. Accordingly,using Natural Variability, breeders combine most useful genes for Yield,Quality, Uniformity, Hardiness, Resistance. The sources of resistancegenes include native or foreign Varieties, Heirloom Varieties, WildPlant Relatives, and Induced Mutations, e.g., treating plant materialwith mutagenic agents. Using the present invention, plant breeders areprovided with a new tool to induce mutations. Accordingly, one skilledin the art can analyze the genome of sources of resistance genes, and inVarieties having desired characteristics or traits employ the presentinvention to induce the rise of resistance genes, with more precisionthan previous mutagenic agents and hence accelerate and improve plantbreeding programs.

Aside from the plants otherwise discussed herein and above, engineeredplants modified by the effector protein and suitable guide, and progenythereof, as provided. These may include disease or drought resistantcrops, such as wheat, barley, rice, soybean or corn; plants modified toremove or reduce the ability to self-pollinate (but which can instead,optionally, hybridise instead); and allergenic foods such as peanuts andnuts where the immunogenic proteins have been disabled, destroyed ordisrupted by targeting via a effector protein and suitable guide.

Therapeutic Treatment

The system of the invention can be applied in areas of former RNAcutting technologies, without undue experimentation, from thisdisclosure, including therapeutic, assay and other applications, becausethe present application provides the foundation for informed engineeringof the system. The present invention provides for therapeutic treatmentof a disease caused by overexpression of RNA, toxic RNA and/or mutatedRNA (such as, for example, splicing defects or truncations). Expressionof the toxic RNA may be associated with formation of nuclear inclusionsand late-onset degenerative changes in brain, heart or skeletal muscle.In the best studied example, myotonic dystrophy, it appears that themain pathogenic effect of the toxic RNA is to sequester binding proteinsand compromise the regulation of alternative splicing (Hum. Mol. Genet.(2006) 15 (suppl 2): R162-R169). Myotonic dystrophy [dystrophiamyotonica (DM)] is of particular interest to geneticists because itproduces an extremely wide range of clinical features. A partial listingwould include muscle wasting, cataracts, insulin resistance, testicularatrophy, slowing of cardiac conduction, cutaneous tumors and effects oncognition. The classical form of DM, which is now called DM type 1(DM1), is caused by an expansion of CTG repeats in the 3′-untranslatedregion (UTR) of DMPK, a gene encoding a cytosolic protein kinase.

The below table presents a list of exons shown to have misregulatedalternative splicing in DM1 skeletal muscle, heart or brain.

Tissue/gene Target Reference Skeletal muscle ALP ex 5a, 5b Lin X., etal. Failure of MBNL1-dependent postnatal splicing transitions inmyotonic dystrophy. Hum. Mol. Genet 2006;15:2087- 2097 CAPN3 ex 16 LinX., et al. Failure of MBNL1-dependent postnatal splicing transitions inmyotonic dystrophy. Hum. Mol. Genet 2006;15:2087- 2097 CLCN1 int 2, ex7a, 8a Mankodi A., et al. Expanded CUG repeats trigger aberrant splicingof ClC-1 chloride channel pre-mRNA and hyperexcitability of skeletalmuscle in myotonic dystrophy. Mol. Cell 2002;10:35-44 Charlet-B N., etal. Loss of the muscle-specific chloride channel in type 1 myotonicdystrophy due to misregulated alternative splicing. Mol. Cell2002;10:45-53 FHOS ex 11a Lin X., et al. Failure of MBNL1-dependentpostnatal splicing transitions in myotonic dystrophy. Hum. Mol. Genet2006;15:2087- 2097 GFAT1 ex 10 Lin X., et al. Failure of MBNL1-dependentpostnatal splicing transitions in myotonic dystrophy. Hum. Mol. Genet2006;15:2087- 2097 IR ex 11 Savkur R.S., et al. Aberrant regulation ofinsulin receptor alternative splicing is associated with insulinresistance in myotonic dystrophy. Nat. Genet. 2001;29:40-47 MBNL1 ex 7Lin X., et al. Failure of MBNL1-dependent postnatal splicing transitionsin myotonic dystrophy. Hum. Mol. Genet 2006;15:2087- 2097 MBNL2 ex 7 LinX., et al. Failure of MBNL1-dependent postnatal splicing transitions inmyotonic dystrophy. Hum. Mol. Genet 2006;15:2087- 2097 MTMR1 ex 2.1, 2.2Buj-Bello A., et al. Muscle-specific alternative splicing ofmyotubularin-related 1 gene is impaired in DM1 muscle cells. Hum. Mol.Genet. 2002;11:2297-2307 NRAP ex 12 Lin X., et al. Failure ofMBNL1-dependent postnatal splicing transitions in myotonic dystrophy.Hum. Mol. Genet 2006;15:2087- 2097 RYR1 ex 70 Kimura T., et al. AlteredmRNA splicing of the skeletal muscle ryanodine receptor andsarcoplasmic/endoplasmic reticulum Ca2+- ATPase in myotonic dystrophytype 1. Hum. Mol. Genet. 2005;14:2189-2200 SERCA1 ex 22 Kimura T., etal. Altered mRNA splicing of the skeletal muscle ryanodine receptor andsarcoplasmic/endoplasmic reticulum Ca2+- ATPase in myotonic dystrophytype 1. Hum. Mol. Genet. 2005;14:2189-2200 Lin X., et al. Failure ofMBNL1-dependent postnatal splicing transitions in myotonic dystrophy.Hum. Mol. Genet 2006;15:2087- 2097 z-Titin ex Zr4, Zr5 Lin X., et al.Failure of MBNL1-dependent postnatal splicing transitions in myotonicdystrophy. Hum. Mol. Genet 2006;15:2087- 2097 m-Titin M-line ex5 Lin X.,et al. Failure of MBNL1-dependent postnatal splicing transitions inmyotonic dystrophy. Hum. Mol. Genet 2006;15:2087- 2097 TNNT3 fetal exKanadia R.N. et al. A muscleblind knockout model for myotonic dystrophy.Science 2003;302:1978-1980 ZASP ex 11 Lin X., et al. Failure ofMBNL1-dependent postnatal splicing transitions in myotonic dystrophy.Hum. Mol. Genet 2006;15:2087- 2097 Heart TNNT2 ex 5 Philips A.V., et al.Disruption of splicing regulated by a CUG- binding protein in myotonicdystrophy. Science 1998;280:737-741 ZASP ex 11 Mankodi A. et al. NuclearRNA foci in the heart in myotonic dystrophy. Circ. Res.2005;97:1152-1155 m-Titin M-line ex 5 Mankodi A. et al. Nuclear RNA fociin the heart in myotonic dystrophy. Circ. Res. 2005;97:1152-1155 KCNAB1ex 2 Mankodi A. et al. Nuclear RNA foci in the heart in myotonicdystrophy. Circ. Res. 2005;97:1152-1155 ALP ex 5 (Mankodi A., et al.Nuclear RNA foci in the heart in myotonic dystrophy. Circ. Res.2005;97:1152-1155 Brain TAU ex 2, ex 10 Sergeant N., et al.Dysregulation of human brain microtubule- associated tau mRNA maturationin myotonic dystrophy type 1. Hum. Mol. Genet. 2001;10:2143-2155 JiangH., et al. Myotonic dystrophy type 1 associated with nuclear foci ofmutant RNA, sequestration of muscleblind proteins, and deregulatedalternative splicing in neurons. Hum. Mol. Genet. 2004;13:3079-3088 APPex 7 Jiang H., et al. Myotonic dystrophy type 1 associated with nuclearfoci of mutant RNA, sequestration of muscleblind proteins, andderegulated alternative splicing in neurons. Hum. Mol. Genet.2004;13:3079-3088 NMDAR1 ex 5 Jiang H., et al. Myotonic dystrophy type 1associated with nuclear foci of mutant RNA, sequestration of muscleblindproteins, and deregulated alternative splicing in neurons. Hum. Mol.Genet. 2004;13:3079-3088

The enzymes of the present invention may target overexpressed RNA ortoxic RNA, such as for example, the DMPK gene or any of the misregulatedalternative splicing in DM1 skeletal muscle, heart or brain in, forexample, the above table.

The enzymes of the present invention may also target trans-actingmutations affecting RNA-dependent functions that cause disease(summarized in Cell. 2009 Feb. 20; 136(4): 777-793) as indicated in thebelow table.

DISEASE GENE/MUTATION FUNCTION Prader Willi syndrome SNORD116 ribosomebiogenesis Spinal muscular atrophy (SMA) SMN2 splicing Dyskeratosiscongenita (X-linked) DKC1 telomerase/translation Dyskeratosis congenitaTERC telomerase (autosomal dominant) Dyskeratosis congenita TERTtelomerase (autosomal dominant) Diamond-Blackfan anemia RPS19, RPS24ribosome biogenesis Shwachman-Diamond syndrome SBDS ribosome biogenesisTreacher-Collins syndrome TCOF1 ribosome biogenesis Prostate cancerSNHG5 ribosome biogenesis Myotonic dystrophy, type 1 (DM1) DMPK proteinkinase (RNA gain-of-function) Myotonic dystrophy type 2 (DM2) ZNF9 RNAbinding (RNA gain-of-function) Spinocerebellar ataxia 8 (SCA8)ATXN8/ATXN8OS unknown/noncoding (RNA gain-of-function) RNA Huntington'sdisease-like 2 (HDL2) JPH3 ion channel function (RNA gain-of-function)Fragile X-associated tremor ataxia FMR1 translation/mRNA syndrome(FXTAS) (RNA gain-of-function) localization Fragile X syndrome FMR1translation/mRNA localization X-linked mental retardation UPF3Btranslation/nonsense mediated decay Oculopharyngeal muscular dystrophyPABPN1 3′ end formation (OPMD) Human pigmentary genodermatosis DSRADediting Retinitis pigmentosa PRPF31 splicing Retinitis pigmentosa PRPF8splicing Retinitis pigmentosa HPRP3 splicing Retinitis pigmentosa PAP1splicing Cartilage-hair hypoplasia (recessive) RMRP splicing Autism7q22-q33 locus breakpoint noncoding RNA Beckwith-Wiedemann syndrome H19noncoding RNA (BWS) Charcot-Marie-Tooth (CMT) Disease GRS translationCharcot-Marie-Tooth (CMT) Disease YRS translation Amyotrophic lateralsclerosis (ALS) TARDBP splicing, transcription Leukoencephalopathy withvanishing EIF2B1 translation white matter Wolcott-Rallison syndromeEIF2AK3 translation (protease) Mitochondrial myopathy and PUS1translation sideroblastic anemia (MLASA) Encephalomyopathy andhypertrophic TSFM translation cardiomyopathy (mitochondrial) Hereditaryspastic paraplegia SPG7 ribosome biogenesis Leukoencephalopathy DARS2translation (mitochondrial) Susceptibility to diabetes mellitus LARS2translation (mitochondrial) Deafness MTRNR1 ribosome biogenesis(mitochondrial) MELAS syndrome, deafness MTRNR2 ribosome biogenesis(mitochondrial) Cancer SFRS1 splicing, translation, export Cancer RBM5splicing Multiple disorders mitochondrial tRNA translation mutations(mitochondrial) Cancer miR-17-92 cluster RNA interference CancermiR-372/miR-373 RNA interference

The enzyme of the present invention may also be used in the treatment ofvarious tauopathies, including primary and secondary tauopathies, suchas primary age-related tauopathy (PART)/Neurofibrillarytangle-predominant senile dementia, with NFTs similar to AD, but withoutplaques, dementia pugilistica (chronic traumatic encephalopathy),progressive supranuclear palsy, corticobasal degeneration,frontotemporal dementia and parkinsonism linked to chromosome 17,lytico-Bodig disease (Parkinson-dementia complex of Guam), gangliogliomaand gangliocytoma, meningioangiomatosis, postencephalitic parkinsonism,subacute sclerosing panencephalitis, as well as lead encephalopathy,tuberous sclerosis, Hallervorden-Spatz disease, and lipofuscinosis,alzheimers disease. The enzymes of the present invention may also targetmutations disrupting the cis-acting splicing code cause splicing defectsand disease (summarized in Cell. 2009 Feb. 20; 136(4): 777-793). Themotor neuron degenerative disease SMA results from deletion of the SMN1gene. The remaining SMN2 gene has a C->T substitution in exon 7 thatinactivates an exonic splicing enhancer (ESE), and creates an exonicsplicing silencer (ESS), leading to exon 7 skipping and a truncatedprotein (SMNΔ7). A T->A substitution in exon 31 of the dystrophin genesimultaneously creates a premature termination codon (STOP) and an ESS,leading to exon 31 skipping. This mutation causes a mild form of DMDbecause the mRNA lacking exon 31 produces a partially functionalprotein. Mutations within and downstream of exon 10 of the MAPT geneencoding the tau protein affect splicing regulatory elements and disruptthe normal 1:1 ratio of mRNAs including or excluding exon 10. Thisresults in a perturbed balance between tau proteins containing eitherfour or three microtubule-binding domains (4R-tau and 3R-tau,respectively), causing the neuropathological disorder FTDP-17. Theexample shown is the N279K mutation which enhances an ESE functionpromoting exon 10 inclusion and shifting the balance toward increased4R-tau. Polymorphic (UG)m(U)n tracts within the 3′ splice site of theCFTR gene exon 9 influence the extent of exon 9 inclusion and the levelof full-length functional protein, modifying the severity of cysticfibrosis (CF) caused by a mutation elsewhere in the CFTR gene.

The innate immune system detects viral infection primarily byrecognizing viral nucleic acids inside an infected cell, referred to asDNA or RNA sensing. In vitro RNA sensing assays can be used to detectspecific RNA substrates. The RNA targeting effector protein can forinstance be used for RNA-based sensing in living cells. Examples ofapplications are diagnostics by sensing of, for examples,disease-specific RNAs.

The RNA targeting effector protein of the invention can further be usedfor antiviral activity, in particular against RNA viruses. The effectorprotein can be targeted to the viral RNA using a suitable guide RNAselective for a selected viral RNA sequence. In particular, the effectorprotein may be an active nuclease that cleaves RNA, such as singlestranded RNA. provided is therefore the use of an RNA targeting effectorprotein of the invention as an antiviral agent.

Therapeutic dosages of the enzyme system of the present invention totarget RNA the above-referenced RNAs are contemplated to be about 0.1 toabout 2 mg/kg the dosages may be administered sequentially with amonitored response, and repeated dosages if necessary, up to about 7 to10 doses per patient. Advantageously, samples are collected from eachpatient during the treatment regimen to ascertain the effectiveness oftreatment. For example, RNA samples may be isolated and quantified todetermine if expression is reduced or ameliorated. Such a diagnostic iswithin the purview of one of skill in the art.

With respect to general information on CRISPR-Cas Systems, mention ismade of the following (also hereby incorporated herein by reference):

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Epub 2015 Sep. 16.-   Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas    System, Zetsche et al., Cell 163, 759-71 (Sep. 25, 2015).-   Discovery and Functional Characterization of Diverse Class 2    CRISPR-Cas Systems, Shmakov et al., Molecular Cell, 60(3), 385-397    doi: 10.1016/j.molcel.2015.10.008 Epub Oct. 22, 2015.-   Rationally engineered Cas9 nucleases with improved specificity,    Slaymaker et al., Science 2016 Jan. 1, 351(6268): 84-88 doi:    10.1126/science.aad5227. Epub 2015 Dec. 1. [Epub ahead of print].    each of which is incorporated herein by reference, may be considered    in the practice of the instant invention, and discussed briefly    below:    -   Cong et al. engineered type II CRISPR-Cas systems for use in        eukaryotic cells based on both Streptococcus thermophilus Cas9        and also Streptococcus pyogenes Cas9 and demonstrated that Cas9        nucleases can be directed by short RNAs to induce precise        cleavage of DNA in human and mouse cells. Their study further        showed that Cas9 as converted into a nicking enzyme can be used        to facilitate homology-directed repair in eukaryotic cells with        minimal mutagenic activity. Additionally, their study        demonstrated that multiple guide sequences can be encoded into a        single CRISPR array to enable simultaneous editing of several at        endogenous genomic loci sites within the mammalian genome,        demonstrating easy programmability and wide applicability of the        RNA-guided nuclease technology. This ability to use RNA to        program sequence specific DNA cleavage in cells defined a new        class of genome engineering tools. These studies further showed        that other CRISPR loci are likely to be transplantable into        mammalian cells and can also mediate mammalian genome cleavage.        Importantly, it can be envisaged that several aspects of the        CRISPR-Cas system can be further improved to increase its        efficiency and versatility.    -   Jiang et al. used the clustered, regularly interspaced, short        palindromic repeats (CRISPR)-associated Cas9 endonuclease        complexed with dual-RNAs to introduce precise mutations in the        genomes of Streptococcus pneumoniae and Escherichia coli. The        approach relied on dual-RNA:Cas9-directed cleavage at the        targeted genomic site to kill unmutated cells and circumvents        the need for selectable markers or counter-selection systems.        The study reported reprogramming dual-RNA:Cas9 specificity by        changing the sequence of short CRISPR RNA (crRNA) to make        single- and multinucleotide changes carried on editing        templates. The study showed that simultaneous use of two crRNAs        enabled multiplex mutagenesis. Furthermore, when the approach        was used in combination with recombineering, in S. pneumoniae,        nearly 100% of cells that were recovered using the described        approach contained the desired mutation, and in E. coli, 65%        that were recovered contained the mutation.    -   Wang et al. (2013) used the CRISPR/Cas system for the one-step        generation of mice carrying mutations in multiple genes which        were traditionally generated in multiple steps by sequential        recombination in embryonic stem cells and/or time-consuming        intercrossing of mice with a single mutation. The CRISPR/Cas        system will greatly accelerate the in vivo study of functionally        redundant genes and of epistatic gene interactions.    -   Konermann et al. (2013) addressed the need in the art for        versatile and robust technologies that enable optical and        chemical modulation of DNA-binding domains based CRISPR Cas9        enzyme and also Transcriptional Activator Like Effectors    -   Ran et al. (2013-A) described an approach that combined a Cas9        nickase mutant with paired guide RNAs to introduce targeted        double-strand breaks. This addresses the issue of the Cas9        nuclease from the microbial CRISPR-Cas system being targeted to        specific genomic loci by a guide sequence, which can tolerate        certain mismatches to the DNA target and thereby promote        undesired off-target mutagenesis. Because individual nicks in        the genome are repaired with high fidelity, simultaneous nicking        via appropriately offset guide RNAs is required for        double-stranded breaks and extends the number of specifically        recognized bases for target cleavage. The authors demonstrated        that using paired nicking can reduce off-target activity by 50-        to 1,500-fold in cell lines and to facilitate gene knockout in        mouse zygotes without sacrificing on-target cleavage efficiency.        This versatile strategy enables a wide variety of genome editing        applications that require high specificity.    -   Hsu et al. (2013) characterized SpCas9 targeting specificity in        human cells to inform the selection of target sites and avoid        off-target effects. The study evaluated >700 guide RNA variants        and SpCas9-induced indel mutation levels at >100 predicted        genomic off-target loci in 293T and 293FT cells. The authors        that SpCas9 tolerates mismatches between guide RNA and target        DNA at different positions in a sequence-dependent manner,        sensitive to the number, position and distribution of        mismatches. The authors further showed that SpCas9-mediated        cleavage is unaffected by DNA methylation and that the dosage of        SpCas9 and sgRNA can be titrated to minimize off-target        modification. Additionally, to facilitate mammalian genome        engineering applications, the authors reported providing a        web-based software tool to guide the selection and validation of        target sequences as well as off-target analyses.    -   Ran et al. (2013-B) described a set of tools for Cas9-mediated        genome editing via non-homologous end joining (NHEJ) or        homology-directed repair (HDR) in mammalian cells, as well as        generation of modified cell lines for downstream functional        studies. To minimize off-target cleavage, the authors further        described a double-nicking strategy using the Cas9 nickase        mutant with paired guide RNAs. The protocol provided by the        authors experimentally derived guidelines for the selection of        target sites, evaluation of cleavage efficiency and analysis of        off-target activity. The studies showed that beginning with        target design, gene modifications can be achieved within as        little as 1-2 weeks, and modified clonal cell lines can be        derived within 2-3 weeks.    -   Shalem et al. described a new way to interrogate gene function        on a genome-wide scale. Their studies showed that delivery of a        genome-scale CRISPR-Cas9 knockout (GeCKO) library targeted        18,080 genes with 64,751 unique guide sequences enabled both        negative and positive selection screening in human cells. First,        the authors showed use of the GeCKO library to identify genes        essential for cell viability in cancer and pluripotent stem        cells. Next, in a melanoma model, the authors screened for genes        whose loss is involved in resistance to vemurafenib, a        therapeutic that inhibits mutant protein kinase BRAF. Their        studies showed that the highest-ranking candidates included        previously validated genes NF1 and MED12 as well as novel hits        NF2, CUL3, TADA2B, and TADA1. The authors observed a high level        of consistency between independent guide RNAs targeting the same        gene and a high rate of hit confirmation, and thus demonstrated        the promise of genome-scale screening with Cas9.    -   Nishimasu et al. reported the crystal structure of Streptococcus        pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 A°        resolution. The structure revealed a bilobed architecture        composed of target recognition and nuclease lobes, accommodating        the sgRNA:DNA heteroduplex in a positively charged groove at        their interface. Whereas the recognition lobe is essential for        binding sgRNA and DNA, the nuclease lobe contains the HNH and        RuvC nuclease domains, which are properly positioned for        cleavage of the complementary and non-complementary strands of        the target DNA, respectively. The nuclease lobe also contains a        carboxyl-terminal domain responsible for the interaction with        the protospacer adjacent motif (PAM). This high-resolution        structure and accompanying functional analyses have revealed the        molecular mechanism of RNA-guided DNA targeting by Cas9, thus        paving the way for the rational design of new, versatile        genome-editing technologies.    -   Wu et al. mapped genome-wide binding sites of a catalytically        inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with        single guide RNAs (sgRNAs) in mouse embryonic stem cells        (mESCs). The authors showed that each of the four sgRNAs tested        targets dCas9 to between tens and thousands of genomic sites,        frequently characterized by a 5-nucleotide seed region in the        sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin        inaccessibility decreases dCas9 binding to other sites with        matching seed sequences; thus 70% of off-target sites are        associated with genes. The authors showed that targeted        sequencing of 295 dCas9 binding sites in mESCs transfected with        catalytically active Cas9 identified only one site mutated above        background levels. The authors proposed a two-state model for        Cas9 binding and cleavage, in which a seed match triggers        binding but extensive pairing with target DNA is required for        cleavage.    -   Platt et al. established a Cre-dependent Cas9 knockin mouse. The        authors demonstrated in vivo as well as ex vivo genome editing        using adeno-associated virus (AAV)-, lentivirus-, or        particle-mediated delivery of guide RNA in neurons, immune        cells, and endothelial cells.    -   Hsu et al. (2014) is a review article that discusses generally        CRISPR-Cas9 history from yogurt to genome editing, including        genetic screening of cells.    -   Wang et al. (2014) relates to a pooled, loss-of-function genetic        screening approach suitable for both positive and negative        selection that uses a genome-scale lentiviral single guide RNA        (sgRNA) library.    -   Doench et al. created a pool of sgRNAs, tiling across all        possible target sites of a panel of six endogenous mouse and        three endogenous human genes and quantitatively assessed their        ability to produce null alleles of their target gene by antibody        staining and flow cytometry. The authors showed that        optimization of the PAM improved activity and also provided an        on-line tool for designing sgRNAs.    -   Swiech et al. demonstrate that AAV-mediated SpCas9 genome        editing can enable reverse genetic studies of gene function in        the brain.    -   Konermann et al. (2015) discusses the ability to attach multiple        effector domains, e.g., transcriptional activator, functional        and epigenomic regulators at appropriate positions on the guide        such as stem or tetraloop with and without linkers.    -   Zetsche et al. demonstrates that the Cas9 enzyme can be split        into two and hence the assembly of Cas9 for activation can be        controlled.    -   Chen et al. relates to multiplex screening by demonstrating that        a genome-wide in vivo CRISPR-Cas9 screen in mice reveals genes        regulating lung metastasis.    -   Ran et al. (2015) relates to SaCas9 and its ability to edit        genomes and demonstrates that one cannot extrapolate from        biochemical assays. Shalem et al. (2015) described ways in which        catalytically inactive Cas9 (dCas9) fusions are used to        synthetically repress (CRISPRi) or activate (CRISPRa)        expression, showing. advances using Cas9 for genome-scale        screens, including arrayed and pooled screens, knockout        approaches that inactivate genomic loci and strategies that        modulate transcriptional activity.    -   End Edits    -   Shalem et al. (2015) described ways in which catalytically        inactive Cas9 (dCas9) fusions are used to synthetically repress        (CRISPRi) or activate (CRISPRa) expression, showing. advances        using Cas9 for genome-scale screens, including arrayed and        pooled screens, knockout approaches that inactivate genomic loci        and strategies that modulate transcriptional activity.    -   Xu et al. (2015) assessed the DNA sequence features that        contribute to single guide RNA (sgRNA) efficiency in        CRISPR-based screens. The authors explored efficiency of        CRISPR/Cas9 knockout and nucleotide preference at the cleavage        site. The authors also found that the sequence preference for        CRISPRi/a is substantially different from that for CRISPR/Cas9        knockout.    -   Parnas et al. (2015) introduced genome-wide pooled CRISPR-Cas9        libraries into dendritic cells (DCs) to identify genes that        control the induction of tumor necrosis factor (Tnf) by        bacterial lipopolysaccharide (LPS). Known regulators of Tlr4        signaling and previously unknown candidates were identified and        classified into three functional modules with distinct effects        on the canonical responses to LPS.    -   Ramanan et al (2015) demonstrated cleavage of viral episomal DNA        (cccDNA) in infected cells. The HBV genome exists in the nuclei        of infected hepatocytes as a 3.2 kb double-stranded episomal DNA        species called covalently closed circular DNA (cccDNA), which is        a key component in the HBV life cycle whose replication is not        inhibited by current therapies. The authors showed that sgRNAs        specifically targeting highly conserved regions of HBV robustly        suppresses viral replication and depleted cccDNA.    -   Nishimasu et al. (2015) reported the crystal structures of        SaCas9 in complex with a single guide RNA (sgRNA) and its        double-stranded DNA targets, containing the 5′-TTGAAT-3′ PAM and        the 5′-TTGGGT-3′ PAM. A structural comparison of SaCas9 with        SpCas9 highlighted both structural conservation and divergence,        explaining their distinct PAM specificities and orthologous        sgRNA recognition.    -   Canver et al. (2015) demonstrated a CRISPR-Cas9-based functional        investigation of non-coding genomic elements. The authors we        developed pooled CRISPR-Cas9 guide RNA libraries to perform in        situ saturating mutagenesis of the human and mouse BCL11A        enhancers which revealed critical features of the enhancers.    -   Zetsche et al. (2015) reported characterization of Cpf1, a class        2 CRISPR nuclease from Francisella novicida U112 having features        distinct from Cas9. Cpf1 is a single RNA-guided endonuclease        lacking tracrRNA, utilizes a T-rich protospacer-adjacent motif,        and cleaves DNA via a staggered DNA double-stranded break.    -   Shmakov et al. (2015) reported three distinct Class 2 CRISPR-Cas        systems. Two system CRISPR enzymes (C2c1 and C2c3) contain        RuvC-like endonuclease domains distantly related to Cpf1. Unlike        Cpf1, C2c1 depends on both crRNA and tracrRNA for DNA cleavage.        The third enzyme (C2c2) contains two predicted HEPN RNase        domains and is tracrRNA independent.    -   Slaymaker et al (2016) reported the use of structure-guided        protein engineering to improve the specificity of Streptococcus        pyogenes Cas9 (SpCas9). The authors developed “enhanced        specificity” SpCas9 (eSpCas9) variants which maintained robust        on-target cleavage with reduced off-target effects.

Also, “Dimeric CRISPR RNA-guided FokI nucleases for highly specificgenome editing”, Shengdar Q. Tsai, Nicolas Wyvekens, Cyd Khayter,Jennifer A. Foden, Vishal Thapar, Deepak Reyon, Mathew J. Goodwin,Martin J. Aryee, J. Keith Joung Nature Biotechnology 32(6): 569-77(2014), relates to dimeric RNA-guided FokI Nucleases that recognizeextended sequences and can edit endogenous genes with high efficienciesin human cells.

With respect to general information on CRISPR-Cas Systems, componentsthereof, and delivery of such components, including methods, materials,delivery vehicles, vectors, particles, AAV, and making and usingthereof, including as to amounts and formulations, all useful in thepractice of the instant invention, reference is made to: U.S. Pat. Nos.8,999,641, 8,993,233, 8,945,839, 8,932,814, 8,906,616, 8,895,308,8,889,418, 8,889,356, 8,871,445, 8,865,406, 8,795,965, 8,771,945 and8,697,359; US Patent Publications US 2014-0310830 (U.S. application Ser.No. 14/105,031), US 2014-0287938 A1 (U.S. application Ser. No.14/213,991), US 2014-0273234 A1 (U.S. application Ser. No. 14/293,674),US2014-0273232 A1 (U.S. application Ser. No. 14/290,575), US2014-0273231 (U.S. application Ser. No. 14/259,420), US 2014-0256046 A1(U.S. application Ser. No. 14/226,274), US 2014-0248702 A1 (U.S.application Ser. No. 14/258,458), US 2014-0242700 A1 (U.S. applicationSer. No. 14/222,930), US 2014-0242699 A1 (U.S. application Ser. No.14/183,512), US 2014-0242664 A1 (U.S. application Ser. No. 14/104,990),US 2014-0234972 A1 (U.S. application Ser. No. 14/183,471), US2014-0227787 A1 (U.S. application Ser. No. 14/256,912), US 2014-0189896A1 (U.S. application Ser. No. 14/105,035), US 2014-0186958 (U.S.application Ser. No. 14/105,017), US 2014-0186919 A1 (U.S. applicationSer. No. 14/104,977), US 2014-0186843 A1 (U.S. application Ser. No.14/104,900), US 2014-0179770 A1 (U.S. application Ser. No. 14/104,837)and US 2014-0179006 A1 (U.S. application Ser. No. 14/183,486), US2014-0170753 (U.S. application Ser. No. 14/183,429), US 2015-0184139(U.S. application Ser. No. 14/324,960), Ser. No. 14/054,414; EuropeanPatents EP 2 764 103 (EP13824232.6), EP 2 784 162 (EP14170383.5) and EP2 771 468 (EP13818570.7); and PCT Patent Publications PCT PatentPublications WO 2014/093661 (PCT/US2013/074743), WO 2014/093694(PCT/US2013/074790), WO 2014/093595 (PCT/US2013/074611), WO 2014/093718(PCT/US2013/074825), WO 2014/093709 (PCT/US2013/074812), WO 2014/093622(PCT/US2013/074667), WO 2014/093635 (PCT/US2013/074691), WO 2014/093655(PCT/US2013/074736), WO 2014/093712 (PCT/US2013/074819), WO 2014/093701(PCT/US2013/074800), WO 2014/018423 (PCT/US2013/051418), WO 2014/204723(PCT/US2014/041790), WO 2014/204724 (PCT/US2014/041800), WO 2014/204725(PCT/US2014/041803), WO 2014/204726 (PCT/US2014/041804), WO 2014/204727(PCT/US2014/041806), WO 2014/204728 (PCT/US2014/041808), WO 2014/204729(PCT/US2014/041809), WO 2015/089351 (PCT/US2014/069897), WO 2015/089354(PCT/US2014/069902), WO 2015/089364 (PCT/US2014/069925), WO 2015/089427(PCT/US2014/070068), WO 2015/089462 (PCT/US2014/070127), WO 2015/089419(PCT/US2014/070057), WO 2015/089465 (PCT/US2014/070135), WO 2015/089486(PCT/US2014/070175), PCT/US2015/051691, PCT/US2015/051830. Reference isalso made to U.S. provisional patent applications 61/758,468;61/802,174; 61/806,375; 61/814,263; 61/819,803 and 61/828,130, filed onJan. 30, 2013; Mar. 15, 2013; Mar. 28, 2013; Apr. 20, 2013; May 6, 2013and May 28, 2013 respectively. Reference is also made to U.S.provisional patent application 61/836,123, filed on Jun. 17, 2013.Reference is additionally made to U.S. provisional patent applications61/835,931, 61/835,936, 61/836,127, 61/836,101, 61/836,080 and61/835,973, each filed Jun. 17, 2013. Further reference is made to U.S.provisional patent applications 61/862,468 and 61/862,355 filed on Aug.5, 2013; 61/871,301 filed on Aug. 28, 2013; 61/960,777 filed on Sep. 25,2013 and 61/961,980 filed on Oct. 28, 2013. Reference is yet furthermade to: PCT Patent applications Nos: PCT/US2014/041803,PCT/US2014/041800, PCT/US2014/041809, PCT/US2014/041804 andPCT/US2014/041806, each filed Jun. 10, 2014; PCT/US2014/041808 filedJun. 11, 2014; and PCT/US2014/62558 filed Oct. 28, 2014, and U.S.Provisional Patent Applications Ser. Nos. 61/915,148, 61/915,150,61/915,153, 61/915,203, 61/915,251, 61/915,301, 61/915,267,61/915,260,and 61/915,397, each filed Dec. 12, 2013; 61/757,972 and 61/768,959,filed on Jan. 29, 2013 and Feb. 25, 2013; 61/835,936, 61/836,127,61/836,101, 61/836,080, 61/835,973, and 61/835,931, filed Jun. 17, 2013;62/010,888 and 62/010,879, both filed Jun. 11, 2014; 62/010,329 and62/010,441, each filed Jun. 10, 2014; 61/939,228 and 61/939,242, eachfiled Feb. 12, 2014; 61/980,012, filed Apr. 15, 2014; 62/038,358, filedAug. 17, 2014; 62/054,490, 62/055,484, 62/055,460 and 62/055,487, eachfiled Sep. 25, 2014; and 62/069,243, filed Oct. 27, 2014. Reference ismade to U.S. provisional patent application 61/930,214 filed on Jan. 22,2014.

Mention is also made of U.S. application 62/180,709, filed 17 Jun. 2015,PROTECTED GUIDE RNAS (PGRNAS); U.S. application 62/091,455, filed, 12Dec. 2014, PROTECTED GUIDE RNAS (PGRNAS); U.S. application 62/096,708,24 Dec. 2014, PROTECTED GUIDE RNAS (PGRNAS); U.S. application62/091,462, 12 Dec. 2014, DEAD GUIDES FOR CRISPR TRANSCRIPTION FACTORS;U.S. application 62/096,324, 23 Dec. 2014, 62/180,681, 17 Jun. 2015, and62/237,496, 5 Oct. 2015, DEAD GUIDES FOR CRISPR TRANSCRIPTION FACTORS;U.S. application 62/091,456, 12 Dec. 2014 and 62/180,692, 17 Jun. 2015,ESCORTED AND FUNCTIONALIZED GUIDES FOR CRISPR-CAS SYSTEMS; U.S.application 62/091,461, 12 Dec. 2014, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR GENOMEEDITING AS TO HEMATOPOETIC STEM CELLS (HSCs); U.S. application62/094,903, 19 Dec. 2014, UNBIASED IDENTIFICATION OF DOUBLE-STRANDBREAKS AND GENOMIC REARRANGEMENT BY GENOME-WISE INSERT CAPTURESEQUENCING; U.S. application 62/096,761, 24 Dec. 2014, ENGINEERING OFSYSTEMS, METHODS AND OPTIMIZED ENZYME AND GUIDE SCAFFOLDS FOR SEQUENCEMANIPULATION; U.S. application 62/098,059, 30 Dec. 2014 and 62/181,667,18 Jun. 2015, RNA-TARGETING SYSTEM; U.S. application 62/096,656, 24 Dec.2014 and 62/181,151, 17 Jun. 2015, CRISPR HAVING OR ASSOCIATED WITHDESTABILIZATION DOMAINS; U.S. application 62/096,697, 24 Dec. 2014,CRISPR HAVING OR ASSOCIATED WITH AAV; U.S. application 62/098,158, 30Dec. 2014, ENGINEERED CRISPR COMPLEX INSERTIONAL TARGETING SYSTEMS; U.S.application 62/151,052, 22 Apr. 2015, CELLULAR TARGETING FOREXTRACELLULAR EXOSOMAL REPORTING; U.S. application 62/054,490, 24 Sep.2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CASSYSTEMS AND COMPOSITIONS FOR TARGETING DISORDERS AND DISEASES USINGPARTICLE DELIVERY COMPONENTS; U.S. application 61/939,154, 12 Feb. 2014,SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITHOPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application 62/055,484, 25Sep. 2014, SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATIONWITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application62/087,537, 4 Dec. 2014, SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCEMANIPULATION WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S.application 62/054,651, 24 Sep. 2014, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR MODELINGCOMPETITION OF MULTIPLE CANCER MUTATIONS IN VIVO; U.S. application62/067,886, 23 Oct. 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OFTHE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR MODELING COMPETITION OFMULTIPLE CANCER MUTATIONS IN VIVO; U.S. application 62/054,675, 24 Sep.2014 and 62/181,002, 17 Jun. 2015, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS IN NEURONALCELLS/TISSUES; U.S. application 62/054,528, 24 Sep. 2014, DELIVERY, USEAND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONSIN IMMUNE DISEASES OR DISORDERS; U.S. application 62/055,454, 25 Sep.2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CASSYSTEMS AND COMPOSITIONS FOR TARGETING DISORDERS AND DISEASES USING CELLPENETRATION PEPTIDES (CPP); U.S. application 62/055,460, 25 Sep. 2014,MULTIFUNCTIONAL-CRISPR COMPLEXES AND/OR OPTIMIZED ENZYME LINKEDFUNCTIONAL-CRISPR COMPLEXES; U.S. application 62/087,475, 4 Dec. 2014,FUNCTIONAL SCREENING WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S.application 62/055,487, 25 Sep. 2014, FUNCTIONAL SCREENING WITHOPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application 62/087,546, 4Dec. 2014 and 62/181,687, 18 Jun. 2015, MULTIFUNCTIONAL CRISPR COMPLEXESAND/OR OPTIMIZED ENZYME LINKED FUNCTIONAL-CRISPR COMPLEXES; and U.S.application 62/098,285, 30 Dec. 2014, CRISPR MEDIATED IN VIVO MODELINGAND GENETIC SCREENING OF TUMOR GROWTH AND METASTASIS.

Mention is made of U.S. applications 62/181,659, 18 Jun. 2015 and62/207,318, 19 Aug. 2015, ENGINEERING AND OPTIMIZATION OF SYSTEMS,METHODS, ENZYME AND GUIDE SCAFFOLDS OF CAS9 ORTHOLOGS AND VARIANTS FORSEQUENCE MANIPULATION. Mention is made of U.S. applications 62/181,675,18 Jun. 2015, and Attorney Docket No. 46783.01.2128, filed 22 Oct. 2015,NOVEL CRISPR ENZYMES AND SYSTEMS, U.S. application 62/232,067, 24 Sep.2015, U.S. application 62/205,733, 16 Aug. 2015, U.S. application62/201,542, 5 Aug. 2015, U.S. application 62/193,507, 16 Jul. 2015, andU.S. application 62/181,739, 18 Jun. 2015, each entitled NOVEL CRISPRENZYMES AND SYSTEMS and of U.S. application 62/245,270, 22 Oct. 2015,NOVEL CRISPR ENZYMES AND SYSTEMS. Mention is also made of U.S.application 61/939,256, 12 Feb. 2014, and WO 2015/089473(PCT/US2014/070152), 12 Dec. 2014, each entitled ENGINEERING OF SYSTEMS,METHODS AND OPTIMIZED GUIDE COMPOSITIONS WITH NEW ARCHITECTURES FORSEQUENCE MANIPULATION. Mention is also made of PCT/US2015/045504, 15Aug. 2015, U.S. application 62/180,699, 17 Jun. 2015, and U.S.application 62/038,358, 17 Aug. 2014, each entitled GENOME EDITING USINGCAS9 NICKASES.

Each of these patents, patent publications, and applications, and alldocuments cited therein or during their prosecution (“appln citeddocuments”) and all documents cited or referenced in the appln citeddocuments, together with any instructions, descriptions, productspecifications, and product sheets for any products mentioned therein orin any document therein and incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention. All documents (e.g., these patents, patent publicationsand applications and the appln cited documents) are incorporated hereinby reference to the same extent as if each individual document wasspecifically and individually indicated to be incorporated by reference.

In addition, mention is made of PCT application PCT/US14/70057, AttorneyReference 47627.99.2060 and BI-2013/107 entitled “DELIVERY, USE ANDTHERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FORTARGETING DISORDERS AND DISEASES USING PARTICLE DELIVERY COMPONENTS(claiming priority from one or more or all of U.S. provisional patentapplications 62/054,490, filed Sep. 24, 2014; 62/010,441, filed Jun. 10,2014; and 61/915,118, 61/915,215 and 61/915,148, each filed on Dec. 12,2013) (“the Particle Delivery PCT”), incorporated herein by reference,with respect to a method of preparing an sgRNA-and-Cas9 proteincontaining particle comprising admixing a mixture comprising an sgRNAand Cas9 protein (and optionally HDR template) with a mixture comprisingor consisting essentially of or consisting of surfactant, phospholipid,biodegradable polymer, lipoprotein and alcohol; and particles from sucha process. For example, wherein Cas9 protein and sgRNA were mixedtogether at a suitable, e.g., 3:1 to 1:3 or 2:1 to 1:2 or 1:1 molarratio, at a suitable temperature, e.g., 15-30C, e.g., 20-25C, e.g., roomtemperature, for a suitable time, e.g., 15-45, such as 30 minutes,advantageously in sterile, nuclease free buffer, e.g., 1×PBS.Separately, particle components such as or comprising: a surfactant,e.g., cationic lipid, e.g., 1,2-dioleoyl-3-trimethylammonium-propane(DOTAP); phospholipid, e.g., dimyristoylphosphatidylcholine (DMPC);biodegradable polymer, such as an ethylene-glycol polymer or PEG, and alipoprotein, such as a low-density lipoprotein, e.g., cholesterol weredissolved in an alcohol, advantageously a C₁₋₆ alkyl alcohol, such asmethanol, ethanol, isopropanol, e.g., 100% ethanol. The two solutionswere mixed together to form particles containing the Cas9-sgRNAcomplexes. Accordingly, sgRNA may be pre-complexed with the Cas9protein, before formulating the entire complex in a particle.Formulations may be made with a different molar ratio of differentcomponents known to promote delivery of nucleic acids into cells (e.g.1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC), polyethyleneglycol (PEG), and cholesterol) For example DOTAP:DMPC:PEG:CholesterolMolar Ratios may be DOTAP 100, DMPC 0, PEG 0, Cholesterol 0; or DOTAP90, DMPC 0, PEG 10, Cholesterol 0; or DOTAP 90, DMPC 0, PEG 5,Cholesterol 5. DOTAP 100, DMPC 0, PEG 0, Cholesterol 0. That applicationaccordingly comprehends admixing sgRNA, Cas9 protein and components thatform a particle; as well as particles from such admixing. Aspects of theinstant invention can involve particles; for example, particles using aprocess analogous to that of the Particle Delivery PCT, e.g., byadmixing a mixture comprising sgRNA and/or Cas9 as in the instantinvention and components that form a particle, e.g., as in the ParticleDelivery PCT, to form a particle and particles from such admixing (or,of course, other particles involving sgRNA and/or Cas9 as in the instantinvention).

The present invention will be further illustrated in the followingExamples which are given for illustration purposes only and are notintended to limit the invention in any way.

EXAMPLES Example 1: Origin and Evolution of Adaptive Immunity Systems

Classification and Annotation of CRISPR-Cas Systems in Archaeal andBacterial Genomes.

The CRISPR-Cas loci has more than 50 gene families and there is nostrictly universal genes, fast evolution, extreme diversity of lociarchitecture. Therefore, no single tree feasible and a multi-prongedapproach is needed. So far, there is comprehensive cas geneidentification of 395 profiles for 93 Cas proteins. Classificationincludes signature gene profiles plus signatures of locus architecture

A new classification of CRISPR-Cas systems is proposed in FIG. 1. Class1 includes multisubunit crRNA-effector complexes (Cascade) and Class 2includes Single-subunit crRNA-effector complexes (Cas9-like). FIG. 2provides a molecular organization of CRISPR-Cas. FIG. 3 providesstructures of Type I and III effector complexes: commonarchitecture/common ancestry despite extensive sequence divergence. FIG.4 shows CRISPR-Cas as a RNA recognition motif (RRM)-centered system.FIG. 5 shows Cas1 phylogeny where recombination of adaptation andcrRNA-effector modules show a major aspect of CRISPR-Cas evolution. FIG.6 shows a CRISPR-Cas census, specifically a distribution of CRISPR-Castypes/subtypes among archaea and bacteria.

Cas1 is not always linked to CRISPR-Cas systems, therefore it may bepossible that there are two branches of “solo” Cas1 which suggests theremay be differences in function and origin and possible novel mobileelements (see Makarova, Krupovic, Koonin, Frontiers Genet 2014). Thegenome organization of three casposon families may provide some clues.In addition to Cas1 and PolB, casposons incorporate diverse genesincluding various nucleases (Krupovic et al. BMC Biology 2014). Onefamily has protein-primed polymerase, another family has RNA-primedpolymerase. In addition to diverse Euryarchaeota and Thaumarchaeota,casposons found in several bacteria which suggests horizontal mobility.Casposon Cas1 (transposase/integrase) suggests a basal clade in the Cas1phylogeny.

Bacteria and archae utilize CRISPR for adaptive immunity in procaryotesand eukaryotes via genome manipulation. Cas 1 provides a ready made toolfor genome manipulation. There are similar mechanisms of integration incasposons and CRISPR, specifically replication-dependent acquisition bycopy/paste not cut-and-paste (Krupovic et al. BMC Biology 2014). Cas1 isa bona fide integrase (Nuñez J K, Lee A S, Engelman A, Doudna J A.Integrase-mediated spacer acquisition during CRISPR-Cas adaptiveimmunity. Nature. 2015 Feb. 18). There is similarity between terminalinverted repeats of casposons and CRISPR (Krupovic et al. BMC Biology2014). CRISPR-Cas may have originated from a casposon and an innateimmunity locus (Koonin, Krupovic, Nature Rev Genet, 2015). The evolutionof adaptive immunity systems in prokaryotes and animals may have beenalong parallel courses with transposon integration at innate immunityloci (Koonin, Krupovic, Nature Rev Genet, 2015). RAG1 transposase (thekey enzyme of V(D)J recombination in vertebrates) may have originatedfrom Transib transposons (Kapitonov V V, Jurka J. RAG1 core and V(D)Jrecombination signal sequences were derived from Transib transposons.PLoS Biol. 2005 June; 3(6):e181), however, none of the Transibs encodesRAG2. RAG1 and RAG2 encoding transposons are described in Kapitonov,Koonin, Biol Direct 2015 and Transib transposase phylogeny is presentedin Kapitonov, Koonin, Biol Direct 2015. Defensive DNA elimination inciliates evolved from a PiggyMAc transposon and RNAi, an innate immunesystem (Swart E C, Nowacki M. The eukaryotic way to defend and editgenomes by sRNA-targeted DNA deletion. Ann N Y Acad Sci. 2015).

The relative stability of the classification implies that the mostprevalent variants of CRISPR-Cas systems are already known. However, theexistence of rare, currently unclassifiable variants implies thatadditional types and subtypes remain to be characterized (Makarova etal. 2015. Evolutionary classification of CRISPR-Cas systems and casgenes).

Transposons play a key contribution to the evolution of adaptiveimmunity and other systems involved in DNA manipulation. Class 1CRISPR-Cas originate from transposons but only for an adaptation module.Class 2 CRISPR-Cas have both both adaptation and effector functionswhere modules may have evolved from different transposons.

Example 2: New Predicted Class 2 CRISPR-Cas Systems and Evidence oftheir Independent Origins from Transposable Elements

The CRISPR-Cas systems of bacterial and archaeal adaptive immunity showextreme diversity of protein composition and genomic loci architecture.These systems are broadly divided into two classes, Class 1 withmultisubunit effector complexes and Class 2 with single-subunit effectormodules exemplified by the Cas9 protein (FIGS. 1A and 1B). Applicantsdeveloped a simple computational pipeline (FIG. 7) to leverage theexpanding genomic and metagenomic databases along with our currentunderstanding of CRISPR-Cas systems for prediction of putative new Class2 CRISPR-Cas systems. Analysis of the database of complete bacterialgenomes using this pipeline resulted in the identification of three newvariants, each represented in diverse bacteria and containing cas1 andcas2 genes along with a third gene encoding a large protein predicted tofunction as the effector module. In the first of these loci, theputative effector protein (C2c1p) contains a RuvC-like nuclease domainand resembles the previously described Cpf1 protein, the predictedeffector of Type V CRISPR-Cas systems; accordingly, the new putativesystem is classified as subtype V-B. In depth comparison of proteinsequences suggests that the RuvC-containing effector proteins, Cas9,Cpf1 and C2C1p independently evolved from different groups oftransposon-encoded TnpB proteins. The second group of new putativeCRISPR-Cas loci encompasses a large protein containing two highlydiverged HEPN domains with predicted RNAse activity. Given the noveltyof the predicted effector protein, these loci are classified as new TypeVI CRISPR-Cas that is likely to target mRNA. Together, the results ofthis analysis show that Class2 CRISPR-Cas systems evolved on multiple,independent occasions, by combination of diverse Cas1-Cas2-encodingadaptation modules with effector proteins derived from different mobileelements. This route of evolution most likely produced multiple variantsof Class 2 systems that remain to be discovered.

The CRISPR-Cas adaptive immunity systems are present in ˜45% bacterialand ˜90% archaeal genomes and show extreme diversity of Cas proteincomposition and sequence, and genomic loci architecture. Based on thestructural organization of their crRNA-effector complexes, these systemsare divided into two classes, namely class 1, with multisubunit effectorcomplexes, and class 2, with single subunit effector complexes(Makarova, 2015) (FIGS. 1A and 1B). Class 1 systems are much more commonand diverse than Class 2 systems. Class 1 currently is represented by 12distinct subtypes encoded by numerous archaeal and bacterial genomes,whereas class 2 systems include three subtypes of Type II system and theputative Type V that collectively are found in about 10%0/of sequencedbacterial genomes (with a single archaeal genome encompassing a putativeType system). Class 2 systems typically contain only three or four genesin the cas operon, namely the cas1-cas2 pair of genes that are involvedin adaptation but not in interference, a single multidomain effectorprotein that is responsible for interference but also contributes to thepre-crRNA processing and adaptation, and often a fourth gene withuncharacterized functions that is dispensable in at least some Type IIsystems. In most cases, a CRISPR array and a gene for a distinct RNAspecies known as tracrRNA (trans-encoded small CRISPR RNA) are adjacentto Class 2 cas operons (Chylinski, 2014). The tracrRNA is partiallyhomologous to the repeats within the respective CRISPR array and isessential for the processing of pre-crRNA that is catalyzed by RNAseIII, a ubiquitous bacterial enzyme that is not associated with theCRISPR-cas loci (Deltcheva, 2011) (Chylinski, 2014; Chylinski, 2013).

The Type II multidomain effector protein Cas9 has been functionally andstructurally characterized in exquisite detail. In different bacteria,Cas9 proteins encompass from about 950 to over 1,600 amino acids, suchas between about 950 and 1,400 amino acids, and contain two nucleasedomains, namely a RuvC-like (RNase H fold) and HNH (McrA-like) nucleases(Makarova, 2011). The crystal structure of Cas9 reveals a bilobedorganization of the protein, with distinct target recognition andnuclease lobes, with the latter accommodating both the RuvC and the HNHdomains (Nishimasu, 2014) (Jinek, 2014). Each of the nuclease domains ofCas9 is required for the cleavage of one of the target DNA strands(Jinek, 2012; Sapranauskas, 2011). Recently, Cas9 has been shown tocontribute to all three stages of the CRISPR response, that is not onlytarget DNA cleavage (interference) but also adaptation and pre-crRNAprocessing (Jinek, 2012). More specifically, a distinct domain in thenuclease lobe of Cas9 has been shown to recognize and bind theProtospacer-Associated Motif (PAM) in viral DNA during the adaptationstage (Nishimasu, 2014) (Jinek, 2014)(Heler, 2015; Wei, 2015). At thisstage of the CRISPR response, Cas9 forms a complex with Cas1 and Cas2,the two proteins that are involved in spacer acquisition in allCRISPR-Cas systems (Heler, 2015; Wei, 2015).

The Cas9 protein, combined with tracrRNA, has recently become the keytool for the new generation of genome editing and engineering methods(Gasiunas, 2013; Mali, 2013; Sampson, 2014; Cong, 2015). This utility ofCas9 in genome editing hinges on the fact that in Type II CRISPR-Cassystems, unlike other types of CRISPR-Cas systems, all the activitiesrequired for the target DNA recognition and cleavage are assembledwithin a single, albeit large, multidomain protein. This feature of TypeII systems greatly facilitates the design of efficient tools for genomemanipulation. Importantly, not all variants of Cas9 are equal. Most ofthe work so far has been done with Cas9 from Streptococcus pyogenes butother Cas9 species could offer substantial advantages. As a case inpoint, recent experiments with Cas9 from Staphylococcus aureus that isabout 300 amino acids shorter than the S. pyogenes protein have allowedCas9 packaging into the adeno-associated virus vector, resulting in amajor enhancement of CRISPR-Cas utility for genome editing in vivo (Ran,2015).

Type II CRISPR-Cas systems currently are classified into 3 subtypes(II-A, II-B and II-C) (Makarova, 2011) (Fonfara, 2014; Chylinski, 2013;Chylinski, 2014). In addition to the cas1, cas2 and cas9 genes that areshared by all Type II loci, subtype II-A is characterized by an extragene, csn2, that encodes an inactivated ATPase (Nam, 2011; Koo, 2012;Lee, 2012) that plays a still poorly characterized role in spaceracquisition (Barrangou, 2007; Arslan, 2013) (Heler, 2015). Subtype II-Bsystems lack csn2 but instead contains the cas4 gene that is otherwisetypical of Type I systems and encodes a recB family 5′-3′ exonucleasethat contributes to spacer acquisition by generating recombinogeneci DNAends (Zhang, 2012) (Lemak, 2013; Lemak, 2014). The cas1 and cas2 genesof subtype II-B are most closely related to the respective proteins ofType I CRISPR-Cas systems which implies a recombinant origin of thisType II subtype (Chylinski, 2014).

Subtype II-C CRISPR-Cas systems are the minimal variety that consistsonly of the cas1, cas2 and cas9 genes (Chylinski, 2013; Koonin, 2013;Chylinski, 2014). Notably, however, it has been shown that inCampylobacter jejuni spacer acquisition by the Type II-C systemsrequires the participation of Cas4 encoded by a bacteriophage (Hooton,2014). Another distinct feature of subtype II-C is the formation of someof the crRNAs by transcription involves transcription from internalalternative promoters as opposed to processing observed in all otherexperimentally characterized CRISPR-Cas systems (Zhang, 2013).

Recently, the existence of Type V CRISPR-Cas systems has been predictedby comparative analysis of bacterial genomes. These putative novelCRISPR-Cas systems are represented in several bacterial genomes, inparticular those from the genus Francisella and one archaeon,Methanomethylophilus alvus (Vestergaard, 2014). All putative Type V lociencompass cas1, cas2, a distinct gene denoted cpf1 and a CRISPR array(Schunder, 2013) (Makarova, 2015). Cpf1 is a large protein (about 1300amino acids) that contains a RuvC-like nuclease domain homologous to thecorresponding domain of Cas9 along with a counterpart to thecharacteristic arginine-rich cluster of Cas9. However, Cpf1 lacks theHNH nuclease domain that is present in all Cas9 proteins, and theRuvC-like domain is contiguous in the Cpf1 sequence, in contrast to Cas9where it contains long inserts including the HNH domain (Chylinski,2014; Makarova, 2015). These major differences in the domainarchitectures of Cas9 and Cpf1 suggest that the Cpf1-contaning systemsshould be classified as a new type. The composition of the putative TypeV systems implies that Cpf1 is a single-subunit effector complex, andaccordingly, these systems are assigned to Class 2 CRISPR-Cas. Some ofthe putative Type V loci encode Cas4 and accordingly resemble subtypeII-B loci, whereas others lack Cas4 and thus are analogous to subtypeII-C.

It has been shown that the closest homologs of Cas9 and Cpf1 proteinsare TnpB proteins that are encoded in IS605 family transposons andcontain the RuvC-like nuclease domain as well as a Zn-finger that has acounterpart in Cpf1. In addition, homologs of TnpB have been identifiedthat contain a HNH domain inserted into the RuvC-like domain and showhigh sequence similarity to Cas9. The role of TnpB in transposonsremains uncertain as it has been shown that this protein is not requiredfor transposition.

Given the homology of Cas9 and Cpf1 to transposon-encoded proteins,Applicants hypothesized that Class 2 CRISPR-Cas systems could haveevolved on multiple occasions as a result of recombination between atransposon and a cas1-cas2 locus. Accordingly, Applicants devised asimple computational strategy to identify genomic loci that could becandidates for novel variants of Class 2. Here Applicants describe thefirst application of this approach that resulted in the identificationof three groups of such candidates two of which appear to be distinctsubtypes of Type V whereas the third one seems to qualify at Type VI.The new variants of Class2 CRISPR-Cas systems are of obvious interest aspotential tools for genome editing and expression regulation.

Database Search Strategy for Detection of Candidate Novel Class 2CRISPR-Cas Loci.

Applicants implemented a straightforward computational approach toidentify candidate novel Class 2 CRISPR-Cas systems (FIG. 7. Pipeline).Because the vast majority of the CRISPR-Cas loci encompass a cas1 gene(Makarova, 2011; Makarova, 2015) and the Cas1 sequence is the mosthighly conserved one among all Cas proteins (Takeuchi, 2012), Applicantsreasoned that cas1 is the best possible anchor to identify candidate newloci using translating PSI-BLAST search with Cas1 profiles. Afterdetecting all contigs encoding Cas1 by searching the WGS (whole genomeshotgun) and NT (nucleotide) databases at the NCBI, the protein-codinggenes were predicted using GenemarkS within the 20 KB regions upstreamand downstream of the cas1 gene. These predicted genes were annotatedusing the NCBI Conserved Domain Database (CDD) and Cas protein-specificprofiles (Makarova et al., 2015, Nat Rev Microbiol. 2015, doi:10.1038/nrmicro3569), and CRISPR arrays were predicted using thePILER-CR program. This procedure provided for assignment of the detectedCRISPR-Cas loci to the known subtypes. Partial and/or unclassifiedcandidate CRISPR-Cas loci containing large (>500 aa) proteins wereselected as candidates for novel Class 2 systems given thecharacteristic presence of such large single-subunit effector proteinsin Types II and V systems (Cas9 and Cpf1, respectively). All 63candidate loci detected using these criteria (listed in the Table setforth in FIG. 40A-D) were analyzed on a case by case basis usingPSI-BLAST and HHpred. The protein sequences encoded in the candidateloci were further used as queries to search metagenomic databases foradditional homologs, and long contigs detected in these searches wereanalyzed as indicated above. This analysis pipeline yielded a total of53 novel loci (some of the originally identified 63 candidate loci werediscarded as spurious whereas several incomplete loci that lacked cas1were added) with characteristic features of Class 2 CRISPR-Cas systemsthat could be classified into three distinct groups of loci based on thenature of the predicted effector proteins (see FIGS. 8A and 8B; FIGS. 9,14 and 15; and FIG. 41A-B, 42A-B, and 43A-B). Although bacteriophagesinfecting bacteria that harbor the newly discovered class 2 CRISPR-Cassystems are virtually unknown, for each of these systems, we detectedspacers that matched phages or predicted prophages.

Using the computational strategy, the Applicants realised three newClass 2 CRISPR-Cas systems, namely C2c1 and C2c3, which are classifiedas subtypes of the previously described putative type V, and C2c2, whichthe Applicants assign to a new putative type VI on the strength of thepresence of a novel predicted effector protein. The Applicants presentmultiple lines of evidence that these loci encode functional CRISPR-Cassystems. On the comparative genomic side, we identified phage-specificspacers for each of the three putative novel systems and also showedthat the sets of spacers are completely different in closely relatedbacterial genomes suggestive of active, functioning immunity. Many ofthese new systems occur in bacterial genomes that encompass no otherCRISPR-Cas loci, suggesting that type V and type VI systems can functionautonomously. Furthermore, even when other CRISPR-Cas systems wereidentified in the same genomes, the associated repeat structures wereclearly distinct from those in types V and VI, suggestive of independentfunctionality.

Putative Type V-B System.

The first group of candidate loci, provisionally denoted C2c1 (Class 2candidate 1), is represented in bacterial genomes from four major taxa,including Bacilli, Verrucomicrobia, alpha-proteobacteria anddelta-proteobacteria (FIG. 8A-B “Organization of complete loci of Class2 systems”; FIG. 41A-B). All C2c1 loci encode a Cas1-Cas4 fusion, Cas2,and the large protein that Applicants denote C2c1p, and typically, areadjacent to a CRISPR array (FIG. 9, C2c1 neighborhoods; FIG. 41A-B). Inthe phylogenetic tree of Cas1, the respective Cas1 proteins cluster withType I-U system (FIGS. 10A and 10B, FIG. 10C-1-W, Cas1 tree), the onlyone in which the Cas1-Cas4 fusion is found. The lengths of the C2c1pproteins identified herein range from about 1100 to about 1500 aminoacids, for example may consist of approximately 1200 amino acids, andHHpred search detected significant similarity between the C-terminalportion of the C2c1p proteins and a subset of TnpB proteins encoded intransposons of the IS605 family (FIGS. 13A-1-A-2 and 13C-1-C-2). Incontrast, no significant similarity was detected between C2c1p and Cas9or Cpf1 that are similar to other groups of TnpB proteins (Chylinski,2014) (Makarova, 2015; Makarova, 2015). Thus, the domain architecture ofC2c1p is similar to that of Cpf1 and distinct from that of Cas9 (FIG.13A-1-A-2) although all three Cas proteins seem to have evolved from theTnpB family (FIG. 11 “Domain organization of class 2 families”; FIG.13A-1-A-2). The N-terminal region of C2c1p shows no significantsimilarity to other proteins. Secondary structure prediction indicatesthat this region adopts mostly alpha-helical conformation. The twosegments of similarity with TnpB encompass the three catalytic motifs ofthe RuvC-like nuclease, with the diagnostic D..E..D signature ofcatalytic amino acid residues (Aravind et al., 2000, Nucleic Acids Res,vol. 28, 3417-3432) (FIG. 12-1-12-2, “TnpB homology regions in Class 2proteins”); the region corresponding to the bridge helix (also known asarginine-rich cluster) that in Cas9 protein is involved incrRNA-binding; and a small region that appears to be the counterpart tothe Zn finger of TnpB (however, the Zn-binding cysteine residues aremissing in the majority of C2c1 proteins indicating that such proteinsdo not bind zinc; moreover, C2c1 contain multiple insertions anddeletions in this region suggestive of functional divergence (FIG.13A-1-A-2, FIG. 13D-1-H-2, FIG. 13I-1-I-4). The conservation of thecatalytic residues (FIG. 13A-1-A-2) strongly suggests that the RuvChomology domains of all these proteins are active nucleases. TheN-terminal regions of C2c1 show no significant similarity to any knownproteins. Secondary structure predictions indicate that the N-terminalregions of C2c1 proteins adopt a mixed α/β conformation (FIG. 13D-1-H-2,FIG. 13I-1-I-4). The similarity of the domain architectures of C2c1p andCpf1 suggests that the C2c1 loci are best classified as Subtype V-B inwhich case the Cpf1-encoding loci become Subtype V-A.

Despite similarity of cas1 genes associated with this system, the CRISPRrepeats in the respective arrays are highly heterogeneous although allof them are 36-37 bp long and can be classified as unstructured (foldingenergy, ΔG, is −0.5-4.5 kcal/mole whereas highly palindromic CRISPR haveΔG below −7 kcal/mole). According to the CRISPRmap (Lange, 2013)classification scheme, several of the Subtype V-B repeats share somesequence or structural similarity with Type II repeats (FIG. 41A-M-2).However, most of the repeats could not be classified into the knownsequence or structure families and were variously assigned to 4 of the 6superclasses (FIG. 41A-M-2).

Considering the possibility that the putative Subtype V-B CRISPR-Cassystems are mechanistically analogous to Type II systems, Applicantsattempted to identify the tracrRNA in the respective genomic loci

Comparison of the spacers from the Type V-B CRISPR arrays to thenon-redundant nucleotide sequence database identified several matches tovarious bacterial genomes. In particular, one of the spacers fromAlicyclobacillus acidoterrestris and one of the spacers fromBrevibacillus agri matched uncharacterized genes within predictedprophages integrated into the respective bacterial genomes (FIG. 41A-L).

Putative Type VI Systems.

The second group of candidate CRISPR-Cas loci, denoted C2c2 (Class 2candidate 2), was identified in genomes from 5 major bacterial taxa,including alpha-proteobacteria, Bacilli, Clostridia, Fusobacteria andBacteroidetes (FIG. 8A-B “Organization of complete loci of Class 2systems”; FIG. 42A-B). A number of C2c2 loci encompass cas1 and cas2genes, along with a large protein (C2c2p) that shows no sequencesimilarity to C2c1, Cpf1, or Cas9, and a CRISPR array; however, unlikeC2c1, C2c2p is often encoded next to a CRISPR array but not cas1-cas2(FIG. 15, C2c2 neighborhoods; FIG. 42A-B). Although under ourcomputational strategy, the originally identified C2c2 loci encompassedthe cas1 and cas2 genes, subsequent searches showed that the majority ofsuch loci may consist only of the c2c2 gene and a CRISPR array. Suchapparently incomplete loci could either encode defective CRISPR-Cassystems or might function with the adaptation module encoded elsewherein the genome, as has been observed for some type III systems (Majumdaret al., 2015, RNA, vol. 21, 1147-1158). In the phylogenetic tree ofCas1, the Cas1 proteins from the C2c2 loci are distributed among twoclades. The first clade includes Cas1 from Clostridia and is locatedwithin the Type II subtree along with a small Type III-A branch (FIGS.10A and 10B, FIG. 10C-1-W, Cas1 tree). The second clade consists of Cas1proteins from C2c2 loci of Leptotrichia and is lodged inside a mixedbranch that mostly contains Cas1 proteins from Type III-A CRISPR-Cassystems. Database searches using HHpred and PSI-BLAST detected nosequence similarity between C2c2p and other proteins. However,inspection of multiple alignments of C2c2p protein sequences led to theidentification of two strictly conserved RxxxxH motifs that arecharacteristic of HEPN (Higher Eukaryotes and ProkaryotesNucleotide-binding) domains (Anantharaman et al., 2013, Biol Direct, vol8, 15; Grynberg et al., 2003, Trends in biochemical sciences, vol. 28,224-226) (FIG. 11 and FIG. 13B, FIG. 13J-1-N-4). Secondary structurepredictions indicates that these motifs are located within structuralcontexts compatible with the HEPN domain structure as is the overallsecondary structure prediction for the respective portions of C2c2p. TheHEPN domains are small (˜150 aa) alpha helical domains with diversesequences but highly conserved catalytic motifs that have been shown orpredicted to possess RNAse activity and are often associated withvarious defense systems (Anantharaman, 2013) (FIGS. 13B and 16-1-16-6,HEPN RxxxxH motif in C2c2 family). The sequences of HEPN domains showlittle conservation except for the catalytic RxxxxH motif. While thesequences of the two putative HEPN domains of C2c2 show littlesimilarity to other HEPN domains except for the catalytic RxxxxH motifs,the domain identity is strongly supported by secondary structurepredictions that indicate that each motif is located within compatiblestructural contexts (FIG. 13B, FIG. 13J-1-N-4). Furthermore, thepredicted secondary structure of the entire sequence for each putativedomain is also consistent with the HEPN fold (FIG. 13J-1-N-4). Thus, itappears likely that C2c2p contains two active HEPN domains. The HEPNdomain is not new to CRISPR-Cas systems as it is often associated withthe CARF (CRISPR-Associated Rossmann Fold) domain in Csm6 and Csx1proteins that are present in many Type III CRISPR-Cas systems (Makarova,2014). These proteins do not belong to either the adaptation modules oreffector complexes but are thought to perform some accessory, stilluncharacterized functions in cognate CRISPR, more particularly theyappear to be components of the associated immunity module that ispresent in the majority of CRISPR-Cas systems and is implicated inprogrammed cell death as well as regulatory functions during the CRISPRresponse (Koonin, 2013; Makarova, 2012; Makarova, 2013). However, C2c2pdiffers from Csm6 and Csx1 in that this much larger protein is the onlycommon protein encoded in the C2c2 loci, except for Cas1 and Cas2. Thus,it appears likely that C2c2p is the effector of these putative novelCRISPR-Cas systems and the HEPN domains are the catalytic moietiesthereof. Outside of the predicted HEPN domains, the C2c2p sequenceshowed no detectable similarity to other proteins and is predicted toadopt a mixed alpha/beta secondary structure without discerniblesimilarity to any known protein folds (FIG. 13J-1-N-4).

The CRISPR arrays in the C2c2 loci are highly heterogeneous, with thelength of 35 to 39 bp, and unstructured (folding energy of −0.9 to 4.7kcal/mole). According to CRISPRmap (Lange, 2013), these CRISPR do notbelong to any of the established structural classes and are assigned to3 of the 6 superclasses. Only the CRISPR from Listeria seeligeri wasassigned to the sequence family 24 that is usually associated with TypeII-C systems (FIG. 42A-L).

Spacer analysis of the C2c2 loci identified one 30 nucleotide regionidentical to a genomic sequence from Listeria weihenstephanensis and twoimperfect hits to bacteriophage genomes, in particular, a spacer fromListeria weihenstephanensis matched the tail gene of a Listeriabacteriophage (FIG. 42A-L).

Given the unique predicted effector complex of C2c2, these systems seemto qualify as a putative Type VI CRISPR-Cas. Furthermore, taking intoaccount that all experimentally characterized and enzymatically activeHEPN domains are RNAses, Type VI systems are likely to act at the levelof RNA, such as mRNA.

Putative Type V-C Systems.

The third group of candidate loci includes solely metagenomic sequencesand thus could not be assigned to specific taxa. These loci encompassonly two protein-coding genes that encode Cas1 and a large proteindenoted C2c3 (Class 2 candidate 3) (FIG. 8A “Organization of completeloci of Class 2 systems”; FIG. 14, C2c3 neighbourhoods, FIG. 43A-B). TheC2c3 proteins are in the same size range as Cpf1 and C2c1, and similarlycontain a TnpB-homologous domain at their C-termini which, unlike therespective domain of C2c1, showed a limited but significant similarityto Cpf1 (FIGS. 13A-1-A-2 and 13C-1-C-2). The TnpB homology regions ofC2c3 contain the three catalytic motifs of the RuvC-like nuclease, withthe diagnostic D..E..D triad of catalytic amino acid residues (Aravindet al., 2000, supra), the region corresponding to the bridge helix (alsoknown as the arginine-rich cluster), which is involved in crRNA-bindingin Cas9, and a small region that appears to be the counterpart to the Znfinger of TnpB (the Zn-binding cysteine residues are conserved in C2c3).The conservation of the catalytic residues strongly suggests that theRuvC homology domains of all these proteins are active nucleases. TheN-terminal regions of C2c1 and C2c3 show no significant similarity toeach other or any known proteins. Secondary structure predictionsindicate that the N-terminal regions of C2c3 proteins adopt a mixed α/βconformation. Thus, the overall domain architectures of C2c1 and C2c3,and in particular the organization of the RuvC domain, are similar tothat of Cpf1 but distinct from that of Cas9. This suggests that the C2c1and C2c3 loci are best classified as subtypes V-B (see above) and V-C,respectively, with Cpf1-encoding loci now designated subtype V-A.

Among the c2c3 loci, only one contains a CRISPR array with unusuallyshort, 17-18 nt spacers. The repeats in this array are 25 bp long andappear to be unstructured with folding energy of −1.6 kcal/mol (FIG.43A-F).

Spacers from the only C2c3 contig containing a CRISPR array are tooshort to produce statistically significant hits. Nevertheless, severalmatches to sequences from predicted prophages were identified (FIG.43A-F).

The subsets of the TnpB proteins with significant similarity to the oneknown (Cas9) and three herein disclosed putative Class 2 effectors(Cpf1, C2c1 and C2c3) did not overlap (FIGS. 13A-1-A-2 and 13C-1-C-2).Although the sequence divergence among the TnpB-like domains is too highto allow reliable phylogenetic analysis, these findings suggest that thefour currently identified large effector proteins of Class 2, Cas9,Cpf1, C2c1 and C2c3, have evolved independently from genes of distincttransposable elements.

Although the majority of spacers in the new CRISPR-Cas loci describedherein were not significantly similar to any available sequences, theexistence of spacers matching phage genomes implies that these loci mayencode active, functional adaptive immunity systems. The small fractionof phage-specific spacers is typical of CRISPR-Cas systems and is mostlikely indicative of their dynamic evolution and the small fraction ofvirus diversity that is represented in the current sequence databases.This interpretation is compatible with the observation that closelyrelated bacterial strains encoding homologous CRISPR-Cas loci typicallycontain unrelated collections of spacers, as exemplified by the C2c2loci from Listeria weihenstephanensis and Listeria newyorkensis (FIG.45A-C).

Applicants applied a simple, straightforward computational strategy topredict new Class 2 CRISPR-cas systems. The previously described class 2systems, namely Type II and the putative Type V, consisted of the cas1and cas2 genes (and in some cases also cas4) comprising the adaptationmodule and a single large protein that comprises the effector module.Therefore, Applicants surmised that any genomic locus containing cas1and a large protein could be a potential candidate for a novel Class 2system that merits detailed investigation. Such analysis using sensitivemethods for protein sequence comparison led to the identification ofthree strong candidates two of which are distinct subtypes of thepreviously described putative Type V (subtypes V-B and V-C) whereas thethird one qualifies as a new putative Type VI, on the strength of thepresence of a novel predicted effector protein. Many of these newsystems occur in bacterial genomes that encompass no other CRISPR-Casloci (FIG. 44A-E-2) suggesting that Type V and Type VI systems canfunction autonomously. The herein disclosed candidate loci werevalidated through functional assays which revealed the expression andprocessing of the respective CRISPR arrays, yielding mature crRNAs,identification of putative tracrRNA (where present), demonstration ofinterference when expressed in E. coli, determination of the protospaceradjacent motif (PAM), and interrogation of the minimal componentsnecessary for lysate cleavage.

Type V systems encode predicted effector proteins that resemble Cas9 intheir overall domain architecture, but in contrast to Cas9, theRuvC-like domains of Cpf1, C2c1 and C2c3 are contiguous in the proteinsequence, lacking the inserts characteristic of Cas9, particularly theHNH nuclease domain. The presence of one instead of two nuclease domainsindicates that type V effector proteins mechanistically differ from Cas9in which the HNH and RuvC domains are responsible for the cleavage ofthe complementary and non-complementary strands of the target DNA,respectively (Chen et al., 2014, The Journal of biological chemistry,vol. 289, 13284-13294; Gasiunas et al., 2012, Proceedings of theNational Academy of Sciences of the United States of America, vol. 109,E2579-2586; Jinek et al., 2012, Science, vol. 337, 816-821). Thepredicted type V effector proteins might form dimers in which the twoRuvC-like domains would cleave the opposite strands of the targetmolecule.

The putative type VI CRISPR-Cas systems seem to rely on a novel effectorprotein that contains two predicted HEPN domains that, similar to thepreviously characterized HEPN domains, could possess RNAse activity,suggesting that type VI systems might target and cleave mRNA.Previously, mRNA targeting has been reported for certain type IIICRISPR-Cas systems (Hale et al., 2014, Genes Dev, vol. 28, 2432-2443;Hale et al., 2009, Cell, vol. 139, 945-956; Peng et al., 2015, Nucleicacids research, vol. 43, 406-417). An alternative possibility is thatC2c2 is the first DNAse in the HEPN superfamily, perhaps with the twoHEPN domains each cleaving one DNA strand. Thus, it might be possible todevelop C2c1 and C2c2 into genome editing tools with different classesof targets.

To validate the functionality of these Class2 CRISPR-Cas systems, theApplicants showed that two C2c1 CRISPR arrays are expressed, processedinto mature crRNAs, and capable of interference when expressed in E.coli. These experiments revealed several characteristics of the C2c1locus including: (i) a 5′ processed DR on the crRNA, (ii) a 5′ PAM, and(iii) the presence of a short RNA with repeat-anti-repeat homology tothe processed 5′ DR, i.e., a putative tracrRNA. The discovery of a 5′processed DR and 5′ PAM supports the scenario in which C2c1 is derivedfrom Class 1 systems because these systems show evidence of 5′ repeatprocessing (type I and III) and a 5′ PAM (type I) (Mojica et al., 2009,Microbiology, vol. 155, 733-740; Makarova et al., 2011, Nat RevMicrobioln vol. 9, 467-477). Notably, the AT-rich PAM identified herefor C2c1 is in contrast to the GC-rich PAMs of the otherwell-characterized Class 2 system (type II). For C2c1 lociexperimentally characterized here, the Applicants identified crRNAs thatare processed to a length that preserves the binding and co-folding withputative tracrRNAs, suggesting that tracrRNAs may be involved in andpossibly required for complex formation. We then used expression of C2c1in a human cell culture to experimentally test that under thosecircumstances a tracrRNA was involved in and necessary for the in vitrocleavage of target DNA by the particular C2c1 nuclease tested.

The Applicants also showed that when the C2c2 locus from L. seeligeri isexpressed in E. coli, it is processed into crRNAs with a 29-nt 5′ DR;similar results were obtained for the C2c2 locus of L. shahii. In thiscase, the degenerate repeat is at the beginning of the array, ratherthan at the end, as is typical for most CRISPR arays, and the array andcas genes are transcribed co-directionally. The Applicants did notdetect the putative tracrRNA in the C2c2 RNA-seq data. However, thepredicted secondary structure of the 29-nt DR shows a stable hairpinhandle which could be potentially important for complex formation withthe C2c2 effector protein.

FIG. 94 demonstrates that processing of the C2c2 array in E colirequires the C2c2 protein, as evaluated with in vitro transcribed spacerarrays incubated with C2c2 protein.

Combined with the results of previous analyses, (Chylinski, 2014;Makarova, 2011), the identification of the novel Class2 CRISPR-Cassystems reveals the dominant theme in the evolution of Class 2CRISPR-Cas systems. The effector proteins of two of the three typeswithin this class appear to have evolved from the pool of transposableelements that encode TnpB proteins containing the RuvC-like domain. Thesequences of the RuvC-like domains of TnpB and the homologous domains ofthe Class 2 effector proteins are too diverged for reliable phylogeneticanalysis. Nevertheless, for Cas9, the effector protein of Type IIsystems, the specific ancestral group seems to be readily identifiable,namely a family of TnpB-like proteins, particularly abundant inCyanobacteria, that show a relatively high sequence similarity to Cas9and share with it the entire domain architecture, namely the RuvC-likeand HNH nuclease domains and the arginine-rich bridge helix (Chylinski,2014) (FIG. 11, FIGS. 13A and 13B, “Domain organization of class 2families”; FIG. 12-1-12-2, FIGS. 13A and 13B, “TnpB homology regions inClass 2 proteins”). Unlike Cas9, it was impossible to trace Cpf1, C2c1,and C2c3 to a specific TnpB family; despite the conservation of allmotifs centered at the catalytic residues of the RuvC-like nucleases,these proteins show only a limited similarity to generic profiles of theTnpB. However, given that C2c1p shows no detectable sequence similaritywith Cpf1, that Cpf1, C2c1, and C2c3 contain distinct insertions betweenthe RuvC-motifs and clearly unrelated N-terminal regions, it appearsmost likely that Cpf1, C2c1, and C2c3 originated independently fromdifferent families within the pool of TnpB-encoding elements (FIG.13C-1-C-2).

It is intriguing that the TnpB proteins seem to be “predesigned” forutilization in Class 2 CRISPR-Cas effector complexes such that theyapparently have been recruited on multiple different occasions.Conceivably, such utility of TnpB proteins has to do with theirpredicted ability to cut a single-stranded DNA while bound to a RNAmolecule via the R-rich bridge helix that in Cas9 has been shown to bindcrRNA (Jinek, 2014; Nishimasu, 2014; Anders et al., 2014, Nature, vol.513, 569-573). The functions of TnpB in the life cycles of therespective transposons are poorly understood. These proteins are notrequired for transposition, and in one case, a TnpB protein has beenshown to down-regulate transposition (Pasternak, 2013) but theirmechanism of action remains unknown. Experimental study of TnpB islikely to shed light on the mechanistic aspects of the Class 2CRISPR-Cas systems. It should be noted that the mechanisms of Cpf1 andC2c1 could be similar to each other but are bound to substantiallydiffer from that of Cas9 because the former two proteins lack the HNHdomain that in Cas9 is responsible for nicking one of the target DNAstrands (Gasiunas, 2012) (Jinek, 2012) (Chen, 2014). Accordingly,exploitation of Cpf1 and C2c1 might bring additional genome editingpossibilities.

In evolutionary terms, it is striking that Class 2 CRISPR-Cas appear tobe completely derived from different transposable elements given therecent evidence on the likely origin of cas1 genes from a distincttransposon family (Koonin, 2015; Krupovic, 2014). Furthermore, thelikely independent origin of the effector proteins from differentfamilies of TnpB, along with the different phylogenetic affinities ofthe respective cas1 proteins, strongly suggest that Class 2 systems haveevolved on multiple occasions through the combination of variousadaptation modules and transposon-derived nucleases giving rise toeffector proteins. This mode of evolution appears to be the ultimatemanifestation of the modularity that is characteristic of CRISPR-Casevolution (Makarova, 2015), with the implication that additionalcombinations of adaptation and effector module are likely to exist innature.

The putative Type VI CRISPR-Cas systems encompass a predicted noveleffector protein that contains two predicted HEPN domain that are likelyto possess RNAse activity. The HEPN domains are not parts of theeffector complexes in other CRISPR-Cas systems but are involved in avariety of defense functions including a predicted ancillary role invarious CRISPR-Cas systems (Anantharaman, 2013) (Makarova, 2015). Thepresence of the HEPN domains as the catalytic moiety of the predictedeffector module implies that the Type VI systems target and cleave mRNA.Previously, mRNA targeting has been reported for certain Type IIICRISPR-Cas systems (Hale, 2014; Hale, 2009) (Peng, 2015). Although HEPNdomains so far have not been detected in bona fide transposableelements, they are characterized by high horizontal mobility and areintegral to mobile elements such as toxin-antitoxin units (Anantharaman,2013). Thus, the putative Type VI systems seem to fit the generalparadigm of the modular evolution of Class 2 CRISPR-Cas from mobilecomponents, and additional variants and new types are expected to bediscovered by analysis of genomic and metagenomics data. Given that theC2c2 protein is unrelated to the other Class 2 effectors (which allcontain RuvC-like domains, even if distantly related ones), thediscovery of type VI can be considered to corroborate the case for theindependent origins of different Class 2 variants.

In view of the emerging scenario of the evolution of Class 2 systemsfrom mobile elements, it seems instructive to examine the overallevolution of CRISPR-Cas loci and in particular the contributions ofmobile elements to this process (FIG. 53). The ancestral adaptiveimmunity system most likely originated via the insertion of a casposon(a Cas1-encoding transposon) adjacent to a locus that encoded aprimitive innate immunity system (Koonin and Krupovic, 2015, Naturereviews Genetics, vol. 16, 184-192; Krupovic et al., 2014, BMC Biology,vol. 12, 36). An additional important contribution was the incorporationof a toxin-antitoxin system that delivered the cas2 gene and might haveoccurred either in the ancestral casposon or in the evolving adaptiveimmunity locus (FIG. 51).

Given the extremely wide spread of Class 1 systems in archaea andbacteria and the proliferation of the ancient RRM (RNA RecognitionMotif) domains in them, there seems to be little doubt that theancestral system was of Class 1 (FIG. 51). Most likely, the ancestralarchitecture resembled the extant type III and in that it encompassed anenzymatically active Cas10 protein (Makarova et al., 2011, Biol Direct,vol. 6, 38; Makarova et al., 2013, Biochem Soc Trans, vol. 41,1392-1400). The Cas10 protein is a homolog of family B DNA polymerasesand nucleotide cyclases of the GGDEF family that shows significantsequence similarity to these enzymes and retains all the catalytic aminoacid residues (Makarova et al., 2011, Biol Direct, vol. 6, 38; Makarovaet al., 2006, Biol Direct, vol. 1, 7). Structural analysis has confirmedthe presence of the polymerase-cyclase-like domain in Cas10 andadditionally revealed a second, degenerate and apparently inactivedomain of this family (Khachatryan et al., 2015, Phys Rev Lett, vol.114, 051801; Shao et al., 2013, Structure, vol. 21, 376-384; Zhu and Ye,2012, FEBS Lett, vol. 586, 939-945). The exact nature of the catalyticactivity of Cas10 remains unclear but it has been shown that thecatalytic residues of the polymerase-cyclase-like domain are essentialfor the target DNA cleavage (Samai et al., 2015, Cell, vol. 161,1164-1174). The Cas8 proteins present in type I CRISPR-Cas systems aresimilar in size to Cas10 and occupy equivalent positions in the effectorcomplexes (Jackson et al., 2014, Science, vol. 345, 1473-1479; Jacksonand Wiedenheft, 2015, Mol Cell, vol. 58, 722-728; Staals et al., 2014,Molecular cell, vol. 56, 518-530), suggestive of an evolutionaryrelationship between the large subunits of the type III and type Ieffector complexes. More specifically, the Cas8 proteins that havehighly diverged in sequence between type I subtypes could becatalytically inactive derivatives of Cas10 (Makarova et al., 2011, BiolDirect, vol. 6, 38; Makarova et al., 2015). This scenario suggests aplausible directionality of evolution, from type III-like ancestralClass 1 system to the type I systems. The divergence of the type III andtype I systems could have been precipitated by the acquisition of theCas3 helicase by the emerging type I (FIG. 53). The different types andsubtypes of Class 2 then evolved via multiple substitutions of the geneblock encoding the Class 1 effector complexes via insertion oftransposable elements encoding various nucleases (FIG. 53). Thisparticular directionality of evolution follows from the observation thatthe adaptation modules of different Class 2 variants derive fromdifferent Class 1 types (FIGS. 10A and 10B).

The Class 2 CRISPR-Cas systems appear to have been completely derivedfrom different mobile elements. Specifically, there seem to have been atleast two (in subtype V-C) but typically, three or, in the case of typeII, even four mobile element contributors: (i) the ancestral casposon,(ii) the toxin-antitoxin module that gave rise to Cas2, (iii) atransposable element, in many cases a TnpB-encoding one, that was theancestor of the Class 2 effector complex, and (iv) in the case of typeII, the HNH nuclease could have been donated to the ancestral transposonby a group I or group II self-splicing intron (Stoddard, 2005, Q RevBiophys, vol. 38, 49-95) (FIG. 53). The putative type V-C loci describedhere encode the ultimate minimalistic CRISPR-Cas system, the onlycurrently identified one that lacks Cas2; conceivably, the highlydiverged subtype V-C Cas1 proteins are capable of forming the adaptationcomplex on their own, without the accessory Cas2 subunit. The multipleoriginations of Class 2 systems from mobile elements present theultimate manifestation of the modularity that is characteristic of theevolution of CRISPR-Cas (Makarova et al., 2015).

The demonstration that different varieties of Class 2 CRISPR-Cas systemsindependently evolved from different transposable elements implies thatadditional variants and new types remain to be identified. Although mostif not all of the new CRISPR-Cas systems are expected to be rare, theycould employ novel strategies and molecular mechanisms and could providea major resource for new, versatile applications in genome engineeringand biotechnology.

Modular evolution is a key feature of CRISPR-Cas systems. This mode ofevolution appears to be most pronounced in Class 2 systems that evolvethrough the combination of adaptation modules from various otherCRISPR-Cas systems with effector proteins that seem to be recruited frommobile elements on multiple independent occasions. Given the extremediversity of mobile elements in bacteria, it appears likely thateffector modules of Class 2 CRISPR-Cas systems are highly diverse aswell. Here Applicants employed a simple computational approach todelineate three new variants of CRISPR-Cas systems but many more arelikely to exist bacterial genomes that have not yet been sequenced.Although most if not all of these new CRISPR-Cas systems are expected tobe rare, they could employ novel strategies and molecular mechanisms andwould provide a major resource for new applications in genomeengineering and biotechnology.

TBLASTN program with the E-value cut-off of 0.01 and low complexityfiltering turned off parameters was used to search with Cas1 profile(Makarova et al., 2015) as a query against NCBI WGS database. Sequencesof contigs or complete genome partitions where Cas1 hit has beenidentified were retrieved from the same database. The region around theCas1 gene (the region 20 kb from the start of the Cas1 gene and 20 kbfrom the end of the Cas1 gene) was extracted and translated usingGeneMarkS (Besemer et al., 2001, supra). Predicted proteins from eachCas1-encoding region were searched against a collection of profiles fromCDD database (Marchler-Bauer, 2009) and specific Cas protein profiles(Makarova et al., 2015) using the RPS-BLAST program (Marchler-Bauer etal., 2002, Nucleic Acids Res, vol. 30, 281-283). Procedure to identifycompleteness of CRISPR loci and to classify CRISPR-Cas systems into theexisting types and subtypes (Makarova et al., 2015) developed previouslyhas been applied to each locus.

CRISPRmap (Lange, 2013) was used for repeat classification.

Partial and/or unclassified loci that encompassed proteins larger than500 amino acids were analyzed on a case-by-case basis. Specifically,each predicted protein encoded in these loci was searched usingiterative profile searches with the PSI-BLAST (Altschul, 1997), andcomposition based-statistics and low complexity filtering turned off, tosearch for distantly similar sequences against NCBI's non-redundant (NR)protein sequence database. Each identified non-redundant protein wassearched against WGS database using the TBLAST program (Altschul, 1997).The HHpred program was used with default parameters to identify remotesequence similarity (Soding, 2005) using as the queries all proteinsidentified in the BLAST searches. Multiple sequence alignments wereconstructed using MUSCLE (Edgar, 2004) and MAFFT (Katoh and Standley,2013, Mol Biol Evol, vol. 30, 772-780). Phylogenetic analysis wasperformed using the FastTree program with the WAG evolutionary model andthe discrete gamma model with 20 rate categories (Price et al., 2010,PLoS One, vol. 5, e9490). Protein secondary structure was predictedusing Jpred 4 (Drozdetskiy, 2015).

CRISPR repeats were identified using PILER-CR (Edgar, 2007, supra) or,for degenerate repeats, CRISPRfinder (Grissa et al., 2007, Nucleic AcidsRes, vol. 35, W52-57). The Mfold program (Zuker, 2003, Nucleic AcidsRes, vol. 31, 3406-3415) was used to identify the most stable structurefor the repeat sequences.

The spacer sequences were searched against the NCBI nucleotide NR andWGS databases using MEGABLAST (Morgulis et al., 2008, Bioinformatics,vol. 24, 1757-1764) with default parameters except that the word sizewas set at 20.

Chosen Gene Candidates

Gene ID: A; Gene Type: C2C1; Organism: 5. Opitutaceae bacterium TAV5; SpacerLength-mode (range): 34 (33 to 37); DR1:GCCGCAGCGAAUGCCGUUUCACGAAUCGUCAGGCGG; DR2: none; tracrRNA1:GCUGGAGACGUUUUUUGAAACGGCGAGUGCUGCGGAUAGCGAGUUUCUCUUGGGGAGGCGCUCGCGGCCACUUUU;tracrRNA2: none; Protein Sequence:MSLNRIYQGRVAAVETGTALAKGNVEWMPAAGGDEVLWQHHELFQAAINYYLVALLALADKNNPVLGPLISQMDNPQSPYHVWGSFRRQGRQRTGLSQAVAPYITPGNNAPTLDEVFRSILAGNPTDRATLDAALMQLLKACDGAGAIQQEGRSYWPKFCDPDSTANFAGDPAMLRREQHRLLLPQVLHDPAITHDSPALGSFDTYSIATPDTRTPQLTGPKARARLEQAITLWRVRLPESAADFDRLASSLKKIPDDDSRLNLQGYVGSSAKGEVQARLFALLLFRHLERSSFTLGLLRSATPPPKNAETPPPAGVPLPAASAADPVRIARGKRSFVFRAFTSLPCWHGGDNIHPTWKSFDIAAFKYALTVINQIEEKTKERQKECAELETDFDYMHGRLAKIPVKYTTGEAEPPPILANDLRIPLLRELLQNIKVDTALTDGEAVSYGLQRRTIRGFRELRRIWRGHAPAGTVFSSELKEKLAGELRQFQTDNSTTIGSVQLFNELIQNPKYWPIWQAPDVETARQWADAGFADDPLAALVQEAELQEDIDALKAPVKLTPADPEYSRRQYDFNAVSKFGAGSRSANRHEPGQTERGHNTFTTEIAARNAADGNRWRATHVRIHYSAPRLLRDGLRRPDTDGNEALEAVPWLQPMMEALAPLPTLPQDLTGMPVFLMPDVTLSGERRILLNLPVTLEPAALVEQLGNAGRWQNQFFGSREDPFALRWPADGAVKTAKGKTHIPWHQDRDHFTVLGVDLGTRDAGALALLNVTAQKPAKPVHRIIGEADGRTWYASLADARMIRLPGEDARLFVRGKLVQEPYGERGRNASLLEWEDARNIILRLGQNPDELLGADPRRHSYPEINDKLLVALRRAQARLARLQNRSWRLRDLAESDKALDEIHAERAGEKPSPLPPLARDDAIKSTDEALLSQRDIIRRSFVQIANLILPLRGRRWEWRPHVEVPDCHILAQSDPGTDDTKRLVAGQRGISHERIEQIEELRRRCQSLNRALRHKPGERPVLGRPAKGEEIADPCPALLEKINRLRDQRVDQTAHAILAAALGVRLRAPSKDRAERRHRDIHGEYERFRAPADFVVIENLSRYLSSQDRARSENTRLMQWCHRQIVQKLRQLCETYGIPVLAVPAAYSSRFSSRDGSAGFRAVHLTPDHRHRMPWSRILARLKAHEEDGKRLEKTVLDEARAVRGLFDRLDRFNAGHVPGKPWRTLLAPLPGGPVFVPLGDATPMQADLNAAINIALRGIAAPDRHDIHHRLRAENKKRILSLRLGTQREKARWPGGAPAVTLSTPNNGASPEDSDALPERVSNLFVDIAGVANFERVTIEGVSQKFATGRGLWASVKQRAWNRVARLNETVTDNNRNEEEDDIPM Gene ID: B; Gene Type: C2C1; Organism:7. Bacillus thermoamylovorans strain B4166; Spacer Length-mode (range):37 (35-38); DR2: GUCCAAGAAAAAAGAAAUGAUACGAGGCAUUAGCAC;DR2: none; tracrRNA1:CUGGACGAUGUCUCUUUUAUUUCUUUUUUCUUGGAUCUGAGUACGAGCACCCACAUUGGACAUUUCGCAUGGUGGGUGCUCGUACUAUAGGUAAAACAAACCUUUUU; tracrRNA2: none; Protein Sequence:MATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAIYEHHEQDPKNPKKVSKAEIQAELWDFVLKMQKCNSFTHEVDKDVVFNILRELYEELVPSSVEKKGEANQLSNKFLYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEKKKWEEDKKKDPLAKILGKLAEYGLIPLFIPFTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWESWNLKVKEEYEKVEKEHKTLEERIKEDIQAFKSLEQYEKERQEQLLRDTLNTNEYRLSKRGLRGWREIIQKWLKMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPYLYATFCEIDKKKKDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESGGWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGTLGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKFVNFKPKELTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLFFPIKGTELYAVHRASFNIKLPGETLVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITEREKRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKSLSDGRKGLYGISLKNIDEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALKEDRLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPYEERSRFENSKLMKWSRREIPRQVALQGEIYGLQVGEVGAQFSSRFHAKTGSPGIRCSVVTKEKLQDNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDRKLVTTHADINAAQNLQKRFWTRTHGFYKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWGNAGKLKIKKGSSKQSSSELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFGKLERILISKLTNQYSISTIEDDSSKQSMGene ID: C; Gene Type: C2C1; Organism: 9. Bacillus sp. NSP2. 1; Spacer Length-mode (range): 36 (35-42); DR1: GUUCGAAAGCUUAGUGGAAAGCUUCGUGGUUAGCAC;DR2: none; tracrRNA1:CACGGAUAAUCACGACUUUCCACUAAGCUUUCGAAUUUUAUGAUGCGAGCAUCCUCUCAGGUCAAAAAA;tracrRNA2: none; Protein Sequence:MAIRSIKLKLKTHTGPEAQNLRKGIWRTHRLLNEGVAYYMKMLLLFRQESTGERPKEELQEELICHIREQQQRNQADKNTQALPLDKALEALRQLYELLVPSSVGQSGDAQIISRKFLSPLVDPNSEGGKGTSKAGAKPTWQKKKEANDPTWEQDYEKWKKRREEDPTASVITTLEEYGIRPIFPLYTNTVTDIAWLPLQSNQFVRTWDRDMLQQAIERLLSWESWNKRVQEEYAKLKEKMAQLNEQLEGGQEWISLLEQYEENRERELRENMTAANDKYRITKRQMKGWNELYELWSTFPASASHEQYKEALKRVQQRLRGRFGDAHFFQYLMEEKNRLIWKGNPQRIHYFVARNELTKRLEEAKQSATMTLPNARKHPLWVRFDARGGNLQDYYLTAEADKPRSRRFVTFSQLIWPSESGWMEKKDVEVELALSRQFYQQVKLLKNDKGKQKIEFKDKGSGSTFNGHLGGAKLQLERGDLEKEEKNFEDGEIGSVYLNVVIDFEPLQEVKNGRVQAPYGQVLQLIRRPNEFPKVTTYKSEQLVEWIKASPQHSAGVESLASGFRVMSIDLGLRAAAATSIFSVEESSDKNAADFSYWIEGTPLVAVHQRSYMLRLPGEQVEKQVMEKRDERFQLHQRVKFQIRVLAQIMRMANKQYGDRWDELDSLKQAVEQKKSPLDQTDRTFWEGIVCDLTKVLPRNEADWEQAVVQIHRKAEEYVGKAVQAWRKRFAADERKGIAGLSMWNIEELEGLRKLLISWSRRTRNPQEVNRFERGHTSHQRLLTHIQNVKEDRLKQLSHAIVMTALGYVYDERKQEWCAEYPACQVILFENLSQYRSNLDRSTKENSTLMKWAHRSIPKYVHMQAEPYGIQIGDVRAEYSSRFYAKTGTPGIRCKKVRGQDLQGRRFENLQKRLVNEQFLTEEQVKQLRPGDIVPDDSGELFMTLTDGSGSKEVVFLQADINAAHNLQKRFWQRYNELFKVSCRVIVRDEEEYLVPKTKSVQAKLGKGLFVKKSDTAWKDVYVWDSQAKLKGKTTFTEESESPEQLEDFQEIIEEAEEAKGTYRTLFRDPSGVFFPESVWYPQKDFWGEVKRKLYGKLRERFLTKARGene ID: D; Gene Type: C2C2; Organism:4. Lachnospiraceae bacterium NK4A144 G619; Spacer Length-mode (range): 35; DR1:GUUUUGAGAAUAGCCCGACAUAGAGGGCAAUAGAC; DR2:GUUAUGAAAACAGCCCGACAUAGAGGGCAAUAGACA;tracrRNAl: none; tracrRNA2: none; Protein Sequence:MKISKVDHTRMAVAKGNQHRRDEISGILYKDPTKTGSIDFDERFKKLNCSAKILYHVFNGIAEGSNKYKNIVDKVNNNLDRVLFTGKSYDRKSIIDIDTVLRNVEKINAFDRISTEEREQIIDDLLEIQLRKGLRKGKAGLREVLLIGAGVIVRTDKKQEIADFLEILDEDFNKTNQAKNIKLSIENQGLVVSPVSRGEERIFDVSGAQKGKSSKKAQEKEALSAFLLDYADLDKNVRFEYLRKIRRLINLYFYVKNDDVMSLTEIPAEVNLEKDFDIWRDHEQRKEENGDFVGCPDILLADRDVKKSNSKQVKIAERQLRESIREKNIKRYRFSIKTIEKDDGTYFFANKQISVFWIHRIENAVERILGSINDKKLYRLRLGYLGEKVWKDILNFLSIKYIAVGKAVFNFAMDDLQEKDRDIEPGKISENAVNGLTSFDYEQIKADEMLQREVAVNVAFAANNLARVTVDIPQNGEKEDILLWNKSDIKKYKKNSKKGILKSILQFFGGASTWNMKMFEIAYHDQPGDYEENYLYDIIQIIYSLRNKSFHFKTYDHGDKNWNRELIGKMIEHDAERVISVEREKFHSNNLPMFYKDADLKKILDLLYSDYAGRASQVPAFNTVLVRKNFPEFLRKDMGYKVHFNNPEVENQWHSAVYYLYKEIYYNLFLRDKEVKNLFYTSLKNIRSEVSDKKQKLASDDFASRCEEIEDRSLPEICQIIMTEYNAQNFGNRKVKSQRVIEKNKDIFRHYKMLLIKTLAGAFSLYLKQERFAFIGKATPIPYETTDVKNFLPEWKSGMYASFVEEIKNNLDLQEWYIVGRFLNGRMLNQLAGSLRSYIQYAEDIERRAAENRNKLFSKPDEKIEACKKAVRVLDLCIKISTRISAEFTDYFDSEDDYADYLEKYLKYQDDAIKELSGSSYAALDHFCNKDDLKFDIYVNAGQKPILQRNIVMAKLFGPDNILSEVMEKVTESAIREYYDYLKKVSGYRVRGKCSTEKEQEDLLKFQRLKNAVEFRDVTEYAEVINELLGQLISWSYLRERDLLYFQLGFHYMCLKNKSFKPAEYVDIRRNNGTIIHNAILYQIVSMYINGLDFYSCDKEGKTLKPIETGKGVGSKIGQFIKYSQYLYNDPSYKLEIYNAGLEVFENIDEHDNITDLRKYVDHFKYYAYGNKMSLLDLYSEFFDRFFTYDMKYQKNVVNVLENILLRHFVIFYPKFGSGKKDVGIRDCKKERAQIEISEQSLTSEDFMFKLDDKAGEEAKKFPARDERYLQTIAKLLYYPNEIEDMNRFMKKGETINKKVQFNRKKKITRKQKNNSSNEVLSSTMGYLFKNIKLGene ID: E; Gene Type: C2C2; Organism:8. Listeria seeligeri serovar 1/2b str. SLCC3954; Spacer Length-mode (range):30; DR1: GUUUUAGUCCUCUUUCAUAUAGAGGUAGUCUCUUAC; DR2: none; tracrRNA1:AUGAAAAGAGGACUAAAACUGAAAGAGGACUAAAACACCAGAUGUGGAUAACUAUAUUAGUGGCUAUUAAAAAUUCGUCGAUAUUAGAGAGGAAACUUU; tracrRNA2: none; Protein Sequence:MWISIKTLIHHLGVLFFCDYMYNRREKKIIEVKTMRITKVEVDRKKVLISRDKNGGKLVYENEMQDNTEQIMHEIKKSSFYKSVVNKTICRPEQKQMKKLVHGLLQENSQEKIKVSDVTKLNISNFLNHRFKKSLYYFPENSPDKSEEYRIEINLSQLLEDSLKKQQGTFICWESFSKDMELYINWAENYISSKTKLIKKSIRNNRIQSTESRSGQLMDRYMKDILNKNKPFDIQSVSEKYQLEKLTSALKATFKEAKKNDKEINYKLKSTLQNHERQIIEELKENSELNQFNIEIRKHLETYFPIKKTNRKVGDIRNLEIGEIQKIVNHRLKNKIVQRILQEGKLASYEIESTVNSNSLQKIKIEEAFALKFINACLFASNNLRNMVYPVCKKDILMIGEFKNSFKEIKHKKFIRQWSQFFSQEITVDDIELASWGLRGAIAPIRNEIIHLKKHSWKKFFNNPTFKVKKSKIINGKTKDVTSEFLYKETLFKDYFYSELDSVPELIINKMESSKILDYYSSDQLNQVFTIPNFELSLLTSAVPFAPSFKRVYLKGFDYQNQDEAQPDYNLKLNIYNEKAFNSEAFQAQYSLFKMVYYQVFLPQFTTNNDLFKSSVDFILTLNKERKGYAKAFQDIRKMNKDEKPSEYMSYIQSQLMLYQKKQEEKEKINHFEKFINQVFIKGFNSFIEKNRLTYICHPTKNTVPENDNIEIPFHTDMDDSNIAFWLMCKLLDAKQLSELRNEMIKFSCSLQSTEEISTFTKAREVIGLALLNGEKGCNDWKELFDDKEAWKKNMSLYVSEELLQSLPYTQEDGQTPVINRSIDLVKKYGTETILEKLFSSSDDYKVSAKDIAKLHEYDVTEKIAQQESLHKQWIEKPGLARDSAWTKKYQNVINDISNYQWAKTKVELTQVRHLHQLTIDLLSRLAGYMSIADRDFQFSSNYILERENSEYRVTSWILLSENKNKNKYNDYELYNLKNASIKVSSKNDPQLKVDLKQLRLTLEYLELFDNRLKEKRNNISHFNYLNGQLGNSILELFDDARDVLSYDRKLKNAVSKSLKEILSSHGMEVTFKPLYQTNHHLKIDKLQPKKIHHLGEKSTVSSNQVSNEYCQLVRTLLTMKGene ID: F; Gene Type: C2C2; Organism: 12. Leptotrichia wadei F0279; SpacerLength-mode (range): 31; DR1: GUUUUAGUCCCCUUCGUUUUUGGGGUAGUCUAAAUC;DR2: none; tracrRNA1:GAUUUAGAGCACCCCAAAAGUAAUGAAAAUUUGCAAUUAAAUAAGGAAUAUUAAAAAAAUGUGAUUUUAAAAAAAUUGAAGAAAUUAAAUGAAAAAUUGUCCAAGUAAAAAAA; tracrRNA2:AUUUAGAUUACCCCUUUAAUUUAUUUUACCAUAUUUUUCUCAUAAUGCAAACUAAUAUUCCAAAAUUUUU;Protein Sequence:MGNLFGHKRWYEVRDKKDFKIKRKVKVKRNYDGNKYILNINENNNKEKIDNNKFIRKYINYKKNDNILKEFTRKFHAGNILFKLKGKEGIIRIENNDDFLETEEVVLYIEAYGKSEKLKALGITKKKIIDEAIRQGITKDDKKIEIKRQENEEEIEIDIRDEYTNKTLNDCSIILRIIENDELETKKSIYEIFKNINMSLYKIIEKIIENETEKVFENRYYEEHLREKLLKDDKIDVILTNFMEIREKIKSNLEILGFVKFYLNVGGDKKKSKNKKMLVEKILNINVDLTVEDIADFVIKELEFWNITKRIEKVKKVNNEFLEKRRNRTYIKSYVLLDKHEKFKIERENKKDKIVKFFVENIKNNSIKEKIEKILAEFKIDELIKKLEKELKKGNCDTEIFGIFKKHYKVNFDSKKFSKKSDEEKELYKIIYRYLKGRIEKILVNEQKVRLKKMEKIEIEKILNESILSEKILKRVKQYTLEHIMYLGKLRHNDIDMTTVNTDDFSRLHAKEELDLELITFFASTNMELNKIFSRENINNDENIDFFGGDREKNYVLDKKILNSKIKIIRDLDFIDNKNNITNNFIRKFTKIGTNERNRILHAISKERDLQGTQDDYNKVINIIQNLKISDEEVSKALNLDVVFKDKKNIITKINDIKISEENNNDIKYLPSFSKVLPEILNLYRNNPKNEPFDTIETEKIVLNALIYVNKELYKKLILEDDLEENESKNIFLQELKKTLGNIDEIDENIIENYYKNAQISASKGNNKAIKKYQKKVIECYIGYLRKNYEELFDFSDFKMNIQEIKKQIKDINDNKTYERITVKTSDKTIVINDDFEYIISIFALLNSNAVINKIRNRFFATSVWLNTSEYQNIIDILDEIMQLNTLRNECITENWNLNLEEFIQKMKEIEKDFDDFKIQTKKEIFNNYYEDIKNNILTEFKDDINGCDVLEKKLEKIVIFDDETKFEIDKKSNILQDEQRKLSNINKKDLKKKVDQYIKDKDQEIKSKILCRIIFNSDFLKKYKKEIDNLIEDMESENENKFQEIYYPKERKNELYIYKKNLFLNIGNPNFDKIYGLISNDIKMADAKFLFNIDGKNIRKNKISEIDAILKNLNDKLNGYSKEYKEKYIKKLKENDDFFAKNIQNKNYKSFEKDYNRVSEYKKIRDLVEFNYLNKIESYLIDINWKLAIQMARFERDMHYIVNGLRELGIIKLSGYNTGISRAYPKRNGSDGFYTTTAYYKFFDEESYKKFEKICYGFGIDLSENSEINKPENESIRNYISHFYIVRNPFADYSIAEQIDRVSNLLSYSTRYNNSTYASVFEVFKKDVNLDYDELKKKFKLIGNNDILERLMKPKKVSVLELESYNSDYIKNLIIELLTKIENTNDTLGene ID: G; Gene Type: C2C2; Organism: 14. Leptotrichia shahii DSM 19757 B031;Spacer Length-mode (range): 30 (30-32); DR1:GUUUUAGUCCCCUUCGAUAUUGGGGUGGUCUAUAUC; DR2: none; tracrRNA1:AUUGAUGUGGUAUACUAAAAAUGGAAAAUUGUAUUUUUGAUUAGAAAGAUGUAAAAUUGAUUUAAUUUAAAAAUAUUUUAUUAGAUUAAAGUAGA; tracrRNA2: none; Protein Sequence:MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNNGene ID: H; Gene Type: Cpf1; Organism: Francisella ularensis subsp. novicidaU112; Spacer Length-mode (range): 31; DR1:GUCUAAGAACUUUAAAUAAUUUCUACUGUUGUAGAU; DR2: none; tracrRNA1:AUCUACAAAAUUAUAAACUAAAUAAAGAUUCUUAUAAUAACUUUAUAUAUAAUCGAAAUGUAGAGAAUUUU;tracrRNA2: none; Protein Sequence:MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN

Genes for Synthesis

For genes A through H, the Applicants optimize the genes for humanexpression and append the following DNA sequence to the end of eachgene. Note this DNA sequence contains a stop codon (underlined), so nostop codon is added to the codon optimized gene sequence:

AAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGggatccTACCCATACGATGTTCCAGATTACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCCTAA

For optimization, avoid the following restriction sites: BamHI, EcoRI,HindIII, BsmBI, BsaI, BbsI, AgeI, XhoI, NdeI, NotI, KpnI, BsrGI, SpeI,XbaI, NheI

These genes are cloned into a simple mammalian expression vector:

>AMSLNRIYQGRVAAVETGTALAKGNVEWMPAAGGDEVLWQHHELFQAAINYYLVALLALADKNNPVLGPLISQMDNPQSPYHVWGSFRRQGRQRTGLSQAVAPYITPGNNAPTLDEVFRSILAGNPTDRATLDAALMQLLKACDGAGAIQQEGRSYWPKFCDPDSTANEAGDPAMLRREQHRLLLPQVLHDPAITHDSPALGSFDTYSIATPDTRTPQLTGPKARARLEQAITLWRVRLPESAADFDRLASSLKKIPDDDSRLNLQGYVGSSAKGEVQARLFALLLFRULERSSFTLGLLRSATPPPKNAETPPPAGVPLPAASAADPVRIARGKRSFVFRAFTSLPCWHGGDNIHPTWKSFDIAAFKYALTVINQIEEKTKERQKECAELETDFDYMHGRLAKIPVKYTTGEAEPPPILANDLRIPLLRELLQNIKVDTALTDGEAVSYGLQRRTIRGFRELRRIWRGHAPAGTVFSSELKEKLAGELRQFQTDNSTTIGSVQLFNELIQNPKYWPIWQAPDVETARQWADAGFADDPLAALVQEAELQEDIDALKAPVKLTPADPEYSRRQYDFNAVSKFGAGSRSANRHEPGQTERGHNTFTTEIAARNAADGNRWRATHVRIHYSAPRLLRDGLRRPDTDGNEALEAVPWLQPMMEALAPLPTLPQDLTGMPVFLMPDVTLSGERRILLNLPVTLEPAALVEQLGNAGRWQNQFFGSREDPFALRWPADGAVKTAKGKTHIPWHQDRDHFTVLGVDLGTRDAGALALLNVTAQKPAKPVHRIIGEADGRTWYASLADARMIRLPGEDARLFVRGKLVQEPYGERGRNASLLEWEDARNIILRLGQNPDELLGADPRRHSYPEINDKLLVALRRAQARLARLQNRSWRLRDLAESDKALDEIHAERAGEKPSPLPPLARDDAIKSTDEALLSQRDIIRRSFVQIANLILPLRGRRWEWRPHVEVPDCHILAQSDPGTDDTKRLVAGQRGISHERIEQIEELRRRCQSLNRALRHKPGERPVLGRPAKGEEIADPCPALLEKINRLRDQRVDQTAHAILAAALGVRLRAPSKDRAERRHRDIHGEYERFRAPADFVVIENLSRYLSSQDRARSENTRLMQWCHRQIVQKLRQLCETYGIPVLAVPAAYSSRFSSRDGSAGFRAVHLTPDHRHRMPWSRILARLKAHEEDGKRLEKTVLDEARAVRGLEDRLDRENAGHVPGKPWRTLLAPLPGGPVFVPLGDATPMQADLNAAINIALRGIAAPDRHDIHHRLRAENKKRILSLRLGTQREKARWPGGAPAVTLSTPNNGASPEDSDALPERVSNLFVDIAGVANFERVTIEGVSQKFATGRGLWASVKQRAWNRVARLNETVTDNNRNEEEDDIPM >BMATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAIYEHHEQDPKNPKKVSKAEIQAELWDFVLKMQKCNSFTHEVDKDVVFNILRELYEELVPSSVEKKGEANQLSNKFLYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEKKKWEEDKKKDPLAKILGKLAEYGLIPLFIPFTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWESWNLKVKEEYEKVEKEHKTLEERIKEDIQAFKSLEQYEKERQEQLLRDTLNTNEYRLSKRGLRGWREIIQKWLKMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPYLYATFCEIDKKKKDAKQQATETLADPINHPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESGGWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGTLGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDEPKEVNEKPKELTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLEEPIKGTELYAVHRASFNIKLPGETLVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITEREKRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKSLSDGRKGLYGISLKNIDEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALKEDRLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPYEERSRFENSKLMKWSRREIPRQVALQGEIYGLQVGEVGAQFSSRFHAKTGSPGIRCSVVTKEKLQDNREEKNLQREGRLTLDKIAVLKEGDLYPDKGGEKEISLSKDRKLVTTHADINAAQNLQKRFWTRTHGFYKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWGNAGKLKIKKGSSKQSSSELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVEFGKLERILISKLTNQYSISTIEDDSSKQSM >CMAIRSIKLKLKTHTGPEAQNLRKGIWRTHRLLNEGVAYYMKMLLLFRQESTGERPKEELQEELICHIREQQQRNQADKNTQALPLDKALEALRQLYELLVPSSVGQSGDAQIISRKFLSPLVDPNSEGGKGTSKAGAKPTWQKKKEANDPTWEQDYEKWKKRREEDPTASVITTLEEYGIRPIFPLYTNTVTDIAWLPLQSNQFVRTWDRDMLQQAIERLLSWESWNKRVQEEYAKLKEKMAQLNEQLEGGQEWISLLEQYEENRERELRENMTAANDKYRITKRQMKGWNELYELWSTFPASASHEQYKEALKRVQQRLRGRFGDAHFFQYLMEEKNRLIWKGNPQRIHYFVARNELTKRLEEAKQSATMTLPNARKHPLWVRFDARGGNLQDYYLTAEADKPRSRRFVTFSQLIWPSESGWMEKKDVEVELALSRQFYQQVKLLKNDKGKQKIEFKDKGSGSTFNGHLGGAKLQLERGDLEKEEKNFEDGEIGSVYLNVVIDFEPLQEVKNGRVQAPYGQVLQLIRRPNEFPKVTTYKSEQLVEWIKASPQHSAGVESLASGFRVMSIDLGLRAAAATSIFSVEESSDKNAADFSYWIEGTPLVAVHQRSYMLRLPGEQVEKQVMEKRDERFQLHQRVKFQIRVLAQIMRMANKQYGDRWDELDSLKQAVEQKKSPLDQTDRTFWEGIVCDLTKVLPRNEADWEQAVVQIHRKAEEYVGKAVQAWRKRFAADERKGIAGLSMWNIEELEGLRKLLISWSRRTRNPQEVNRFERGHTSHQRLLTHIQNVKEDRLKQLSHAIVMTALGYVYDERKQEWCAEYPACQVILFENLSQYRSNLDRSTKENSTLMKWAHRSIPKYVHMQAEPYGIQIGDVRAEYSSRFYAKTGTPGIRCKKVRGQDLQGRRFENLQKRLVNEQFLTEEQVKQLRPGDIVPDDSGELFMTLTDGSGSKEVVFLQADINAAHNLQKRFWQRYNELFKVSCRVIVRDEEEYLVPKTKSVQAKLGKGLFVKKSDTAWKDVYVWDSQAKLKGKTTFTEESESPEQLEDFQEIIEEAEEAKGTYRTLFRDPSGVFFPESVWYPQKDFWGEVKRKLYGKLRERFLTKAR >DMKISKVDHTRMAVAKGNQHRRDEISGILYKDPTKTGSIDFDERFKKLNCSAKILYHVENGIAEGSNKYKNIVDKVNNNLDRVLFTGKSYDRKSIIDIDTVLRNVEKINAFDRISTEEREQIIDDLLEIQLRKGLRKGKAGLREVLLIGAGVIVRTDKKQEIADFLEILDEDENKTNQAKNIKLSIENQGLVVSPVSRGEERIFDVSGAQKGKSSKKAQEKEALSAFLLDYADLDKNVRFEYLRKIRRLINLYFYVKNDDVMSLTEIPAEVNLEKDFDIWRDHEQRKEENGDFVGCPDILLADRDVKKSNSKQVKIAERQLRESIREKNIKRYRFSIKTIEKDDGTYFFANKQISVFWIHRIENAVERILGSINDKKLYRLRLGYLGEKVWKDILNFLSIKYIAVGKAVFNFAMDDLQEKDRDIEPGKISENAVNGLTSFDYEQIKADEMLQREVAVNVAFAANNLARVTVDIPQNGEKEDILLWNKSDIKKYKKNSKKGILKSILQFFGGASTWNMKMFEIAYHDQPGDYEENYLYDIIQIIYSLRNKSFHFKTYDHGDKNWNRELIGKMIEHDAERVISVEREKFHSNNLPMFYKDADLKKILDLLYSDYAGRASQVPAENTVLVRKNEPEFLRKDMGYKVHFNNPEVENQWH SAVYYLYKEIYYNLFLRDKEVKNLFYTSLKNIRSEVSDKKQKLASDDFASRCEEIEDRSLPEICQIIMTEYNAQNFGNRKVKSQRVIEKNKDIFRHYKMLLIKTLAGAFSLYLKQERFAFIGKATPIPYETTDVKNFLPEWKSGMYASFVEEIKNNLDLQEWYIVGRFLNGRMLNQLAGSLRSYIQYAEDIERRAAENRNKLFSKPDEKIEACKKAVRVLDLCIKISTRISAEFTDYEDSEDDYADYLEKYLKYQDDAIKELSGSSYAALDHFCNKDDLKFDIYVNAGQKPILQRNIVMAKLFGPDNILSEVMEKVTESAIREYYDYLKKVSGYRVRGKCSTEKEQEDLLKFQRLKNAVEFRDVTEYAEVINELLGQLISWSYLRERDLLYFQLGFHYMCLKNKSFKPAEYVDIRRNNGTIIHNAILYQIVSMYINGLDFYSCDKEGKTLKPIETGKGVGSKIGQFIKYSQYLYNDPSYKLEIYNAGLEVFENIDEHDNITDLRKYVDHFKYYAYGNKMSLLDLYSEFFDRFFTYDMKYQKNVVNVLENILLRHFVIFYPKFGSGKKDVGIRDCKKERAQIEISEQSLTSEDFMFKLDDKAGEEAKKFPARDERYLQTIAKLLYYPNEIEDMNRFMKKGETINKKVQFNRKKKITRKQKNNSSNEVLSSTMGYLFKNIKL >EMWISIKTLIHHLGVLFFCDYMYNRREKKIIEVKTMRITKVEVDRKKVLISRDKNGGKLVYENEMQDNTEQIMEIHKKSSFYKSVVNKTICRPEQKQMKKLVHGLLQENSQEKIKVSDVTKLNISNFLNHRFKKSLYYFPENSPDKSEEYRIEINLSQLLEDSLKKQQGTFICWESFSKDMELYINWAENYISSKTKLIKKSIRNNRIQSTESRSGQLMDRYMKDILNKNKPFDIQSVSEKYQLEKLTSALKATFKEAKKNDKEINYKLKSTLQNHERQIIEELKENSELNQFNIEIRKHLETYFPIKKTNRKVGDIRNLEIGEIQKIVNHRLKNKIVQRILQEGKLASYEIESTVNSNSLQKIKIEEAFALKFINACLFASNNLRNMVYPVCKKDILMIGEFKNSFKEIKHKKFIRQWSQFFSQEITVDDIELASWGLRGAIAPIRNEIIHLKKHSWKKFFNNPTFKVKKSKIINGKTKDVTSEFLYKETLFKDYFYSELDSVPELIINKMESSKILDYYSSDQLNQVFTIPNFELSLLTSAVPFAPSFKRVYLKGFDYQNQDEAQPDYNLKLNIYNEKAFNSEAFQAQYSLFKMVYYQVFLPQFTTNNDLFKSSVDFILTLNKERKGYAKAFQDIRKMNKDEKPSEYMSYIQSQLMLYQKKQEEKEKINHFEKFINQVFIKGFNSFIEKNRLTYICHPTKNTVPENDNIEIPFHTDMDDSNIAFWLMCKLLDAKQLSELRNEMIKFSCSLQSTEEISTFTKAREVIGLALLNGEKGCNDWKELFDDKEAWKKNMSLYVSEELLQSLPYTQEDGQTPVINRSIDLVKKYGTETILEKLFSSSDDYKVSAKDIAKLHEYDVTEKIAQQESLHKQWIEKPGLARDSAWTKKYQNVINDISNYQWAKTKVELTQVRHLHQLTIDLLSRLAGYMSIADRDFQFSSNYILERENSEYRVTSWILLSENKNKNKYNDYELYNLKNASIKVSSKNDPQLKVDLKQLRLTLEYLELFDNRLKEKRNNISHFNYLNGQLGNSILELFDDARDVLSYDRKLKNAVSKSLKEILSSHGMEVTFKPLYQTNHHLKIDKLQPKKIHHLGEKSTVSSNQVSNEYCQLVRTLLTMK >FMKVTKVDGISHKKYIEEGKLVKSTSEENRTSERLSELLSIRLDIYIKNPDNASEEENRIRRENLKKFFSNKVLHLKDSVLYLKNRKEKNAVQDKNYSEEDISEYDLKNKNSFSVLKKILLNEDVNSEELEIFRKDVEAKLNKINSLKYSFEENKANYQKINENNVEKVGGKSKRNIIYDYYRESAKRNDYINNVQEAFDKLYKKEDIEKLFFLIENSKKHEKYKIREYYHKIIGRKNDKENFAKIIYEEIQNVNNIKELIEKIPDMSELKKSQVFYKYYLDKEELNDKNIKYAFCHFVEIEMSQLLKNYVYKRLSNISNDKIKRIFEYQNLKKLIENKLLNKLDTYVRNCGKYNYYLQVGEIATSDFIARNRQNEAFLRNIIGVSSVAYFSLRNILETENENDITGRMRGKTVKNNKGEEKYVSGEVDKIYNENKQNEVKENLKMFYSYDFNMDNKNEIEDFFANIDEAISSIRHGIVHFNLELEGKDIFAFKNIAPSEISKKMFQNEINEKKLKLKIFKQLNSANVFNYYEKDVIIKYLKNTKFNFVNKNIPFVPSFTKLYNKIEDLRNTLKFFWSVPKDKEEKDAQIYLLKNIYYGEFLNKFVKNSKVFFKITNEVIKINKQRNQKTGHYKYQKFENIEKTVPVEYLAIIQSREMINNQDKEEKNTYIDFIQQIFLKGFIDYLNKNNLKYIESNNNNDNNDIFSKIKIKKDNKEKYDKILKNYEKHNRNKEIPHEINEFVREIKLGKILKYTENLNMFYLILKLLNHKELTNLKGSLEKYQSANKEETFSDELELINLLNLDNNRVTEDFELEANEIGKFLDFNENKIKDRKELKKFDTNKIYFDGENIIKHRAFYNIKKYGMLNLLEKIADKAKYKISLKELKEYSNKKNEIEKNYTMQQNLHRKYARPKKDEKFNDEDYKEYEKAIGNIQKYTHLKNKVEFNELNLLQGLLLKILHRLVGYTSIWERDLRFRLKGEFPENHYIEEIFNFDNSKNVKYKSGQIVEKYINFYKELYKDNVEKRSIYSDKKVKKLKQEKKDLYIRNYIAHFNYIPHAEISLLEVLENLRKLLSYDRKLKNAIMKSIVDILKEYGFVATFKIGADKKIEIQTLESEKIVHLKNLKKKKLMTDRNSEELCELVKVMFEYKALE >GMGNLFGHKRWYEVRDKKDFKIKRKVKVKRNYDGNKYILNINENNNKEKIDNNKFIRKYINYKKNDNILKEFTRKFHAGNILFKLKGKEGIIRIENNDDFLETEEVVLYIEAYGKSEKLKALGITKKKIIDEAIRQGITKDDKKIEIKRQENEEEIEIDIRDEYTNKTLNDCSITLRIIENDELETKKSIYEIFKNINMSLYKIIEKIIENETEKVFENRYYEEHLREKLLKDDKIDVILTNFMEIREKIKSNLEILGFVKFYLNVGGDKKKSKNKKMLVEKILNINVDLTVEDIADFVIKELEFWNITKRIEKVKKVNNEFLEKRRNRTYIKSYVLLDKHEKFKIERENKKDKIVKFFVENIKNNSIKEKIEKILAEFKIDELIKKLEKELKKGNCDTEIFGIFKKHYKVNFDSKKFSKKSDEEKELYKIIYRYLKGRIEKILVNEQKVRLKKMEKIEIEKILNESILSEKILKRVKQYTLEHIMYLGKLRHNDIDMTTVNTDDFSRLHAKEELDLELITFFASTNMELNKIFSRENINNDENIDFFGGDREKNYVLDKKILNSKIKIIRDLDFIDNKNNITNNFIRKFTKIGTNERNRILHAISKERDLQGTQDDYNKVINIIQNLKISDEEVSKALNLDVVFKDKKNIITKINDIKISEENNNDIKYLPSFSKVLPEILNLYRNNPKNEPFDTIETEKIVLNALIYVNKELYKKLILEDDLEENESKNIFLQELKKTLGNIDEIDENIIENYYKNAQISASKGNNKAIKKYQKKVIECYIGYLRKNYEELEDESDFKMNIQEIKKQIKDINDNKTYERITVKTSDKTIVINDDEEYIISTEALLNSNAVINKIRNRFFATSVWLNTSEYQNIIDILDEIMQLNTLRNECITENWNLNLEEFIQKMKEIEKDEDDEKIQTKKEIENNYYEDIKNNILTEEKDDINGCDVLEKKLEKIVIEDDETKEEIDKKSNILQDEQRKLSNINKKDLKKKVDQYIKDKDQEIKSKILCRIIENSDFLKKYKKEIDNLIEDMESENENKFQEIYYPKERKNELYIYKKNLELNIGNPNEDKIYGLISNDIKMADAKELENIDGKNIRKNKISEIDAILKNLNDKLNGYSKEYKEKYIKKLKENDDFFAKNIQNKNYKSFEKDYNRVSEYKKIRDLVEFNYLNKIESYLIDINWKLAIQMARFERDMHYIVNGLRELGIIKLSGYNTGISRAYPKRNGSDGEYTTTAYYKFFDEESYKKFEKICYGFGIDLSENSEINKPENESIRNYISHFYIVRNPFADYSIAEQIDRVSNLLSYSTRYNNSTYASVFEVFKKDVNLDYDELKKKFKLIGNNDILERLMKPKKVSVLELESYNSDYIKNLIIELLTKIENTNDTL >HMSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKEKNLENQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKENTIIGGKEVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANEAAIPMIEDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKEKLNEENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVEFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKEEENIEDCRKEIDEYKQSISKHPEWKDFGERESDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRETEDKEFEHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTENIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGEKRGREKVEKQVYQKLEKMLIEKLNYLVEKDNEEDKTGGVLRAYQLTAPEETEKKMGKQTGITYYVPAGETSKICPVTGEVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSEDYKNEGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN

A-locus through G-locus are cloned and inserted into a low-copy plasmid.A vector that does not contain Amp resistance is used.

>A-locusTATCCGGTCGAATCGAGAATGACGACCGCTACGTCTTGGACTACGAAGCCGTGGCCCTTGCCGATGCTCTCGGTGTGGATGTTGCCGACCTGTTCCGCAAGATCGATTGCCCCAAGAACCTGCTGCGCAGGCGGGCAGGGTAGGGGAGCGGTTTCCGGCGGAGATTTTCGGAGGCGCCGGTAACGTTATGTCGGGGAATTTGCTATACATCGACGATAATTAGTTTTGTTGATTCAGGATCGAAATGCGCTCAAACAAAGAACGTTCCGCGTTTCCCTCATGCGCTACTACGCCCACACCGCCATCTTTCGGCACGCAAACAAAGCAGATGGGTTGCCTGTCAATGGGTGATCATTGCCTGAAGTTACCATCCATCAATAATATAAATCATCCTTACTCCGAATGTCCCTCAATCGCATCTATCAAGGCCGCGTGGCGGCCGTCGAAACAGGAACGGCCTTAGCGAAAGGTAATGTCGAATGGATGCCTGCCGCAGGAGGCGACGAAGTTCTCTGGCAGCACCACGAACTTTTCCAAGCTGCCATCAACTACTATCTCGTCGCCCTGCTCGCACTCGCCGACAAAAACAATCCCGTACTTGGCCCGCTGATCAGCCAGATGGATAATCCCCAAAGCCCTTACCATGTCTGGGGAAGTTTCCGCCGCCAAGGACGTCAGCGCACAGGTCTCAGTCAAGCCGTTGCACCTTATATCACGCCGGGCAATAACGCTCCCACCCTTGACGAAGTTTTCCGCTCCATTCTTGCGGGCAACCCAACCGACCGCGCAACTTTGGACGCTGCACTCATGCAATTGCTCAAGGCTTGTGACGGCGCGGGCGCTATCCAGCAGGAAGGTCGTTCCTACTGGCCCAAATTCTGCGATCCTGACTCCACTGCCAACTTCGCGGGAGATCCGGCCATGCTCCGGCGTGAACAACACCGCCTCCTCCTTCCGCAAGTTCTCCACGATCCGGCGATTACTCACGACAGTCCTGCCCTTGGCTCGTTCGACACTTATTCGATTGCTACCCCCGACACCAGAACTCCTCAACTCACCGGCCCCAAGGCACGCGCCCGTCTTGAGCAGGCGATCACCCTCTGGCGCGTCCGTCTTCCCGAATCGGCTGCTGACTTCGATCGCCTTGCCAGTTCCCTCAAAAAAATTCCGGACGACGATTCTCGCCTTAACCTTCAGGGCTACGTCGGCAGCAGTGCGAAAGGCGAAGTTCAGGCCCGTCTTTTCGCCCTTCTGCTATTCCGTCACCTGGAGCGTTCCTCCTTTACGCTTGGCCTTCTCCGTTCCGCCACCCCGCCGCCCAAGAACGCTGAAACACCTCCTCCCGCCGGCGTTCCTTTACCTGCGGCGTCCGCAGCCGATCCGGTGCGGATAGCCCGTGGCAAACGCAGTTTTGTTTTTCGCGCATTCACCAGTCTCCCCTGCTGGCATGGCGGTGATAACATCCATCCCACCTGGAAGTCATTCGACATCGCAGCGTTCAAATATGCCCTCACGGTCATCAACCAGATCGAGGAAAAGACGAAAGAACGCCAAAAAGAATGTGCGGAACTTGAAACTGATTTCGACTACATGCACGGACGGCTCGCCAAGATTCCGGTAAAATACACGACCGGCGAAGCCGAACCGCCCCCCATTCTCGCAAACGATCTCCGCATCCCCCTCCTCCGCGAACTTCTCCAGAATATCAAGGTCGACACCGCACTCACCGATGGCGAAGCCGTCTCCTATGGTCTCCAACGCCGCACCATTCGCGGTTTCCGCGAGCTGCGCCGCATCTGGCGCGGCCATGCCCCCGCTGGCACGGTCTTTTCCAGCGAGTTGAAAGAAAAACTAGCCGGCGAACTCCGCCAGTTCCAGACCGACAACTCCACCACCATCGGCAGCGTCCAACTCTTCAACGAACTCATCCAAAACCCGAAATACTGGCCCATCTGGCAGGCTCCTGACGTCGAAACCGCCCGCCAATGGGCCGATGCCGGTTTTGCCGACGATCCGCTCGCCGCCCTTGTGCAAGAAGCCGAACTCCAGGAAGACATCGACGCCCTCAAGGCTCCAGTCAAACTCACTCCGGCCGATCCTGAGTATTCAAGAAGGCAATACGATTTCAATGCCGTCAGCAAATTCGGGGCCGGCTCCCGCTCCGCCAATCGCCACGAACCCGGGCAGACGGAGCGCGGCCACAACACCTTTACCACCGAAATCGCCGCCCGTAACGCGGCGGACGGGAACCGCTGGCGGGCAACCCACGTCCGCATCCATTACTCCGCTCCCCGCCTTCTTCGTGACGGACTCCGCCGACCTGACACCGACGGCAACGAAGCCCTGGAAGCCGTCCCTTGGCTCCAGCCCATGATGGAAGCCCTCGCCCCTCTCCCGACGCTTCCGCAAGACCTCACAGGCATGCCGGTCTTCCTCATGCCCGACGTCACCCTTTCCGGTGAGCGTCGCATCCTCCTCAATCTTCCTGTCACCCTCGAACCAGCCGCTCTTGTCGAACAACTGGGCAACGCCGGTCGCTGGCAAAACCAGTTCTTCGGCTCCCGCGAAGATCCATTCGCTCTCCGATGGCCCGCCGACGGTGCTGTAAAAACCGCCAAGGGGAAAACCCACATACCTTGGCACCAGGACCGCGATCACTTCACCGTACTCGGCGTGGATCTCGGCACGCGCGATGCCGGGGCGCTCGCTCTTCTCAACGTCACTGCGCAAAAACCGGCCAAGCCGGTCCACCGCATCATTGGTGAGGCCGACGGACGCACCTGGTATGCCAGCCTTGCCGACGCTCGCATGATCCGCCTGCCCGGGGAGGATGCCCGGCTCTTTGTCCGGGGAAAACTCGTTCAGGAACCCTATGGTGAACGCGGGCGAAACGCGTCTCTTCTCGAATGGGAAGACGCCCGCAATATCATCCTTCGCCTTGGCCAAAATCCCGACGAACTCCTCGGCGCCGATCCCCGGCGCCATTCGTATCCGGAAATAAACGATAAACTTCTCGTCGCCCTTCGCCGCGCTCAGGCCCGTCTTGCCCGTCTCCAGAACCGGAGCTGGCGGTTGCGCGACCTTGCAGAATCGGACAAGGCCCTTGATGAAATCCATGCCGAGCGTGCCGGGGAGAAGCCTTCTCCGCTTCCGCCCTTGGCTCGCGACGATGCCATCAAAAGCACCGACGAAGCCCTCCTTTCCCAGCGTGACATCATCCGGCGATCCTTCGTTCAGATCGCCAACTTGATCCTTCCCCTTCGCGGACGCCGATGGGAATGGCGGCCCCATGTCGAGGTCCCGGATTGCCACATCCTTGCGCAGAGCGATCCCGGTACGGATGACACCAAGCGTCTTGTCGCCGGACAACGCGGCATCTCTCACGAGCGTATCGAGCAAATCGAAGAACTCCGTCGTCGCTGCCAATCCCTCAACCGTGCCCTGCGTCACAAACCCGGAGAGCGTCCCGTGCTCGGACGCCCCGCCAAGGGCGAGGAAATCGCCGATCCCTGTCCCGCGCTCCTCGAAAAGATCAACCGTCTCCGGGACCAGCGCGTTGACCAAACCGCGCATGCCATCCTCGCCGCCGCTCTCGGTGTTCGACTCCGCGCCCCCTCAAAAGACCGCGCCGAACGCCGCCATCGCGACATCCATGGCGAATACGAACGCTTTCGTGCGCCCGCTGATTTTGTCGTCATCGAAAACCTCTCCCGTTATCTCAGCTCGCAGGATCGTGCTCGTAGTGAAAACACCCGTCTCATGCAGTGGTGCCATCGCCAGATCGTGCAAAAACTCCGTCAGCTCTGCGAGACCTACGGCATCCCCGTCCTCGCCGTCCCGGCGGCCTACTCATCGCGTTTTTCTTCCCGGGACGGCTCGGCCGGATTCCGGGCCGTCCATCTGACACCGGACCACCGTCACCGGATGCCATGGAGCCGCATCCTCGCCCGCCTCAAGGCCCACGAGGAAGACGGAAAAAGACTCGAAAAGACGGTGCTCGACGAGGCTCGCGCCGTCCGGGGACTCTTTGACCGGCTCGACCGGTTCAACGCCGGGCATGTCCCGGGAAAACCTTGGCGCACGCTCCTCGCGCCGCTCCCCGGCGGCCCTGTGTTTGTCCCCCTCGGGGACGCCACACCCATGCAGGCCGATCTGAACGCCGCCATCAACATCGCCCTCCGGGGCATCGCGGCTCCCGACCGCCACGACATCCATCACCGGCTCCGTGCCGAAAACAAAAAACGCATCCTGAGCTTGCGTCTCGGCACTCAGCGCGAGAAAGCCCGCTGGCCTGGAGGAGCTCCGGCGGTGACACTCTCCACTCCGAACAACGGCGCCTCTCCCGAAGATTCCGATGCGTTGCCCGAACGGGTATCCAACCTGTTTGTGGACATCGCCGGTGTCGCCAACTTCGAGCGAGTCACGATCGAAGGAGTCTCGCAAAAATTCGCCACCGGGCGTGGCCTTTGGGCCTCCGTCAAGCAACGTGCATGGAACCGCGTTGCCAGACTCAACGAGACAGTAACAGATAACAACAGGAACGAAGAGGAGGACGACATTCCGATGTAACCATTGCTTCATTACATCTGAGTCTCCCCTCAATCCCTCTGCCCCATGCGTGATATAACCTCCACCTCATGTCCCGGATCGGCGCCGGCAACCTGTAGTTCCCTTCCATCCTCCAACACTCCCGCAGATCGCGATCCGCTGCCGCCGATGCCGGTGCGCCGCCTTCACAACTATCTCTACTGTCCGCGGCTTTTTTATCTCCAGTGGGTCGAGAATCTCTTTGAGGAAAATGCCGACACCATTGCCGGCAGCGCCGTGCATCGTCACGCCGACAAACCTACGCGTTACGATGATGAAAAAGCCGAGGCACTTCGCACTGGTCTCCCTGAAGGCGCGCACATACGCAGCCTTCGCCTGGAAAACGCCCAACTCGGTCTCGTTGGCGTGGTGGATATCGTGGAGGGAGGCCCCGACGGACTCGAACTCGTCGACTACAAAAAAGGTTCCGCCTTCCGCCTCGACGACGGCACGCTCGCTCCCAAGGAAAACGACACCGTGCAACTTGCCGCCTACGCTCTTCTCCTGGCTGCCGATGGTGCGCGCGTTGCGCCCATGGCGACGGTCTATTACGCTGCCGATCGCCGGCGTGTCACCTTCCCGCTCGATGACGCCCTCTACGCCCGCACCCGTTCCGCCCTCGAAGAGGCCCGCGCCGTTGCAACCTCGGGGCGCATACCTCCGCCGCTCGTCTCTGACGTCCGCTGCCTCCATTGTTCCTCCTATGCGCTTTGCCTTCCCCGCGAGTCCGCCTGGTGGTGCCGCCATCGCAGCACGCCGCGGGGAGCCGGCCACACCCCCATGTTGCCGGGCTTTGAGGATGACGCCGCCGCCATTCACCAAATCTCCGAACCTGACACCGAGCCACCACCCGATCTTGCCAGCCAGCCTCCCCGTCCCCCGCGGCTCGATGGAGAATTGTTGGTTGTCCAGACTCCGGGAGCGATGATCGGACAAAGCGGCGGTGAGTTTACCGTGTCCGTCAAGGGTGAGGTTTTGCGCAAGCTTCCGGTTCATCAACTCCGGGCCATTTACGTTTACGGAGCCGTGCAACTCACGGCGCATGCTGTGCAGACCGCCCTTGAGGAGGATATCGACGTCTCCTATTTTGCGCCCAGCGGCCGCTTTCTTGGCCTCCTCCGCGGCCTGCCCGCATCCGGCGTGGATGCGCGTCTCGGGCAATACACCCTGTTTCGCGAACCCTTTGGCCGTCTCCGTCTCGCCTGCGAGGCGATTCGGGCCAAGATCCATAACCAGCGCGTCCTCCTCATGCGTAACGGCGAGCCCGGGGAGGGCGTCTTGCGCGAACTCGCCCGTCTGCGCGACGCCACCAGTGAGGCGACTTCGCTCGACGAACTCCTCGGCATCGAGGGCATCGCCGCGCATTTCTATTTCCAGTATTTTCCCACCATGCTGAAAGAACGGGCGGCCTGGGCCTTTGATTTTTCCGGACGCAATCGCCGCCCGCCGCGCGACCCGGTCAACGCCCTGCTTTCGTTCGGTTACAGCGTGTTGTCCAAGGAACTTGCCGGCGTCTGCCACGCTGTTGGCCTAGACCCGTTTTTCGGCTTCATGCACCAGCCGCGTTACGGGCGCCCCGCACTCGCTCTCGATCTGATGGAGGAGTTTCGCCCTCTCATCGCCGACAGTGTTGCCCTGAATCTCATCAACCGTGGCGAACTCGACGAAGGGGACTTTATCCGGTCGGCCAATGGCACCGCGCTCAATGATCGGGGCCGCCGGCGTTTTTGGGAGGCATGGTTCCGGCGTCTCGACAGCGAAGTCAGCCATCCTGAATTTGGTTACAAGATGAGCTATCGACGGATGCTTGAAGTGCAGGCGCGCCAGCTATGGCGCTATGTGCGCGGTGACGCCTTCCGCTACCACGGATTCACCACCCGTTGATTCCGATGTCAGATCCCCGCCGCCGTTATCTTGTGTGTTACGACATCGCCAATCCGAAGCGATTGCGCCAAGTGGCCAAGCTGCTGGAGAGCTATGGCACGCGTCTGCAATACTCGGTTTTCGAATGTCCTTTGGACGATCTTCGTCTTGAACAGGCGAAGGCTGATTTGCGCGACACGATTAATGCCGACCAAGACCAGGTGTTATTTGTTTCGCTTGGCCCCGAAGCCAACGATGCCACGTTGATCATCGCCACGCTTGGGCTCCCTTATACCGTGCGCTCGCGAGTGACGATTATCTGACCCATAACCCACGTGTTGAAGAGGCTGAAAACAGACGGACCTCTATGAAGAACAATTGACGTTTTGGCCGAACTCAGCAGACCTTTATGCGGCTAAGGCCAATGATCATCCATCCTACCGCCATTGGGCTGGAGACGTTTTTTGAAACGGCGAGTGCTGCGGATAGCGAGTTTCTCTTGGGGAGGCGCTCGCGGCCACTTTTACAGAGGAGATGTTCGGGCGAACTGGCCGACCTAACAAGGCGTACCCGGCTCAAAATCGAGGCACGCTCGCACGGGATGATGTAATTCGTTGTTTTTCAGCATACCGTGCGAGCACGGGCCGCAGCGAATGCCGTTTCACGAATCGTCAGGCGGCGGGGAGAAGTCATTTAATAAGGCCACTGTTAAAAGCCGCAGCGAATGCCGTTTCACGAATCGTCAGGCGGGCAGTGGATGTTTTTCCATGAGGCGAAGAATTTCATCGCCGCAGTGAATGCCGTTTCACCATTGATGAAGAATGCGAGGTGAAAACAGAGAAATTGGGTCAACTCTATCACTCTTATTCAGCCATCGTTTCAAGAAAGGATACCTCGTATTGGATACAACACAGCTCGTTCGTTCTCTCTACCTCCCTCGACAATCTCAAGGA >B-locusTAATAAAATTGAAATATCACTATGGATTATTGTAATATTACCATAAAGATAGGTGACGTTTTTTTGAAAATTGTAAACCTAATTTGAAGAAAACCAATTAAAAATCGCTTCGGCTTTTTTTTAAGTGCCAGGTAGCATTGATGCTAACCCATGTGTAATAAAGGTTTGTTTTCCTTCGGGGCACGAACACATTATAAGGGAAACCTAAAGATTCCCTTTCTTGTTTAATATTATAACCAGTGAAAATAAGAATAATGCACCTAAAACTAATATACAGAAAATAAGAATTAAAAGTACTAATATATACATCATATGTTATCCTCCAATGCTTTATTTTTTAATAATTGATGTTAGTATTAGTTTTATTTTAATTTCTAAACATAAGAATTTGAAAAGGATGTGTTTATTATGGCGACACGCAGTTTTATTTTAAAAATTGAACCAAATGAAGAAGTTAAAAAGGGATTATGGAAGACGCATGAGGTATTGAATCATGGAATTGCCTACTACATGAATATTCTGAAACTAATTAGACAGGAAGCTATTTATGAACATCATGAACAAGATCCTAAAAATCCGAAAAAAGTTTCAAAAGCAGAAATACAAGCCGAGTTATGGGATTTTGTTTTAAAAATGCAAAAATGTAATAGTTTTACACATGAAGTTGACAAAGATGTTGTTTTTAACATCCTGCGTGAACTATATGAAGAGTTGGTCCCTAGTTCAGTCGAGAAAAAGGGTGAAGCCAATCAATTATCGAATAAGTTTCTGTACCCGCTAGTTGATCCGAACAGTCAAAGTGGGAAAGGGACGGCATCATCCGGACGTAAACCTCGGTGGTATAATTTAAAAATAGCAGGCGACCCATCGTGGGAGGAAGAAAAGAAAAAATGGGAAGAGGATAAAAAGAAAGATCCCCTTGCTAAAATCTTAGGTAAGTTAGCAGAATATGGGCTTATTCCGCTATTTATTCCATTTACTGACAGCAACGAACCAATTGTAAAAGAAATTAAATGGATGGAAAAAAGTCGTAATCAAAGTGTCCGGCGACTTGATAAGGATATGTTTATCCAAGCATTAGAGCGTTTTCTTTCATGGGAAAGCTGGAACCTTAAAGTAAAGGAAGAGTATGAAAAAGTTGAAAAGGAACACAAAACACTAGAGGAAAGGATAAAAGAGGACATTCAAGCATTTAAATCCCTTGAACAATATGAAAAAGAACGGCAGGAGCAACTTCTTAGAGATACATTGAATACAAATGAATACCGATTAAGCAAAAGAGGATTACGTGGTTGGCGTGAAATTATCCAAAAATGGCTAAAGATGGATGAAAATGAACCATCAGAAAAATATTTAGAAGTATTTAAAGATTATCAACGGAAACATCCACGAGAAGCCGGGGACTATTCTGTCTATGAATTTTTAAGCAAGAAAGAAAATCATTTTATTTGGCGAAATCATCCTGAATATCCTTATTTGTATGCTACATTTTGTGAAATTGACAAAAAAAAGAAAGACGCTAAGCAACAGGCAACTTTTACTTTGGCTGACCCGATTAACCATCCGTTATGGGTACGATTTGAAGAAAGAAGCGGTTCGAACTTAAACAAATATCGAATTTTAACAGAGCAATTACACACTGAAAAGTTAAAAAAGAAATTAACAGTTCAACTTGATCGTTTAATTTATCCAACTGAATCCGGCGGTTGGGAGGAAAAAGGTAAAGTAGATATCGTTTTGTTGCCGTCAAGACAATTTTATAATCAAATCTTCCTTGATATAGAAGAAAAGGGGAAACATGCTTTTACTTATAAGGATGAAAGTATTAAATTCCCCCTTAAAGGTACACTTGGTGGTGCAAGAGTGCAGTTTGACCGTGACCATTTGCGGAGATATCCGCATAAAGTAGAATCAGGAAATGTTGGACGGATTTATTTTAACATGACAGTAAATATTGAACCAACTGAGAGCCCTGTTAGTAAGTCTTTGAAAATACATAGGGACGATTTCCCCAAGTTCGTTAATTTTAAACCGAAAGAGCTCACCGAATGGATAAAAGATAGTAAAGGGAAAAAATTAAAAAGTGGTATAGAATCCCTTGAAATTGGTCTACGGGTGATGAGTATCGACTTAGGTCAACGTCAAGCGGCTGCTGCATCGATTTTTGAAGTAGTTGATCAGAAACCGGATATTGAAGGGAAGTTATTTTTTCCAATCAAAGGAACTGAGCTTTATGCTGTTCACCGGGCAAGTTTTAACATTAAATTACCGGGTGAAACATTAGTAAAATCACGGGAAGTATTGCGGAAAGCTCGGGAGGACAACTTAAAATTAATGAATCAAAAGTTAAACTTTCTAAGAAATGTTCTACATTTCCAACAGTTTGAAGATATCACAGAAAGAGAGAAGCGTGTAACTAAATGGATTTCTAGACAAGAAAATAGTGATGTTCCTCTTGTATATCAAGATGAGCTAATTCAAATTCGTGAATTAATGTATAAACCCTATAAAGATTGGGTTGCCTTTTTAAAACAACTCCATAAACGGCTAGAAGTCGAGATTGGCAAAGAGGTTAAGCATTGGCGAAAATCATTAAGTGACGGGAGAAAAGGTCTTTACGGAATCTCCCTAAAAAATATTGATGAAATTGATCGAACAAGGAAATTCCTTTTAAGATGGAGCTTACGTCCAACAGAACCTGGGGAAGTAAGACGCTTGGAACCAGGACAGCGTTTTGCGATTGATCAATTAAACCACCTAAATGCATTAAAAGAAGATCGATTAAAAAAGATGGCAAATACGATTATCATGCATGCCTTAGGTTACTGTTATGATGTAAGAAAGAAAAAGTGGCAGGCAAAAAATCCAGCATGTCAAATTATTTTATTTGAAGATTTATCTAACTACAATCCTTACGAGGAAAGGTCCCGTTTTGAAAACTCAAAACTGATGAAGTGGTCACGGAGAGAAATTCCACGACAAGTCGCCTTACAAGGTGAAATTTACGGATTACAAGTTGGGGAAGTAGGTGCCCAATTCAGTTCAAGATTCCATGCGAAAACCGGGTCGCCGGGAATTCGTTGCAGTGTTGTAACGAAAGAAAAATTGCAGGATAATCGCTTTTTTAAAAATTTACAAAGAGAAGGACGACTTACTCTTGATAAAATCGCAGTTTTAAAAGAAGGAGACTTATATCCAGATAAAGGTGGAGAAAAGTTTATTTCTTTATCAAAGGATCGAAAGTTGGTAACTACGCATGCTGATATTAACGCGGCCCAAAATTTACAGAAGCGTTTTTGGACAAGAACACATGGATTTTATAAAGTTTACTGCAAAGCCTATCAGGTTGATGGACAAACTGTTTATATTCCGGAGAGCAAGGACCAAAAACAAAAAATAATTGAAGAATTTGGGGAAGGCTATTTTATTTTAAAAGATGGTGTATATGAATGGGGTAATGCGGGGAAACTAAAAATTAAAAAAGGTTCCTCTAAACAATCATCGAGTGAATTAGTAGATTCGGACATACTGAAAGATTCATTTGATTTAGCAAGTGAACTTAAGGGAGAGAAACTCATGTTATATCGAGATCCGAGTGGAAACGTATTTCCTTCCGACAAGTGGATGGCAGCAGGAGTATTTTTTGGCAAATTAGAAAGAATATTGATTTCTAAGTTAACAAATCAATACTCAATATCAACAATAGAAGATGATTCTTCAAAACAATCAATGTAAAAGTTTGCCCGTATAAGAACTTAATTAATTAGGATGGTAGGATGTTACTAAATATGTCTGTAGGCATCATTCCTACTATCCGTTTTGTCCGAATATCAGAGCATTAGGTGAGGAATGGTAAGAAAGGAAAATTTATATGAACCAACCGATTCCTATTCGAATGTTAAATGAAATACAATATTGTGAGCGACTTTTTTACTTTATGCATGTCCAAAAGCTATTTGATGAGAATGCAGATACAGTTGAAGGAAGTGCACAGCATGAGCGGGCAGAAAGAAGCAAAAGACCAAGTAAAATGGGACCAAAGGAATTATGGGGTGAGGCGCCAAGAAGTCTTAAGCTTGGTGATGAGCTGTTAAATATTACCGGTGTTCTTGATGCCATAAGTCATGAAGAGAACAGTTGGATCCCGGTTGAATCAAAACACAGTTCCGCACCGGATGGATTGAACCCTTTTAAAGTAGATGGCTTTCTACTTGACGGGTCTGCATGGCCAAACGATCAAATTCAACTTTGTGCACAAGGCTTGCTCTTGAATGCCAATGGATACCCGTGTGATTATGGGTATTTATTTTATCGTGGTAATAAGAAAAAGGTGAAAATTTATTTTACTGAAGATTTAATCGCTGCCACAAAGTACTATATTAAAAAAGCACACGAGATACTAGTATTATCTGGTGATGAATCAGCTATTCCTAAGCCTTTAATTGATTCTAATAAGTGTTTTCGCTGTTCTTTAAACTATATCTGTCTTCCGGATGAAACGAACTATCTATTAGGGGCAAGTTCAACAATTCGTAAAATTGTGCCTTCAAGGACAGATGGTGGCGTTTTATATGTATCAGAGTCTGGTACAAAATTAGGAAAATCGGGTGAGGAGTTAATCATTCAGTATAAAGATGGCCAAAAGCAGGGTGTTCCTATAAAAGATATTATTCAAGTTTCGTTAATTGGAAATGTTCAATGCTCAACGCAATTACTTCATTTTTTAATGCAATCAAATATTCCTGTAAGTTATTTATCATCCCACGGTCGTTTGATTGGTGTCAGTTCATCTTTAGTTACAAAAAATGTTTTAACAAGGCAGCAACAGTTCATTAAATTTACAAATCCTGAGTTTGGACTAAATCTAGCAAAACAAATTGTTTATGCCAAGATTCGAAATCAACGAACTTTACTTAGAAGAAATGGGGGGAGTGAGGTAAAGGAGATTTTAACAGATTTAAAATCTTTAAGTGACAGTGCACTGAACGCAATATCAATAGAACAATTACGGGGTATTGAAGGGATTTCTGCAAAACATTATTTCGCAGGATTTCCGTTTATGTTGAAAAATGAATTACGTGAATTGAATTTAATGAAAGGGCGTAATAGGAGACCGCCAAAAGATCCTGTAAATGTACTTCTTTCTCTTGGTTATACTTTATTGACACGTGATATTCATGCTGCGTGTGGTTCAGTCGGATTGGATCCGATGTTTGGTTGTTACCATCGTCCAGAAGCAGGTCGACCGGCTCTAGTATTAGATGTTATGGAAACATTTCGACCACTTATTGTAGACAGTATTGTCATCCGAGCTTTGAATACGGGTGAAATCTCATTAAAAGATTTTTATATAGGAAAAGATAGTTGTCAATTATTAAAACATGGCCGCGATTCCTTTTTTGCCATTTATGAAAGAAGAATGCATGAAACTATTACCGATCCAATTTTCGGCTATAAGATTAGCTATCGCCGTATGCTCGATTTGCACATTCGAATGCTTGCAAGGTTTATTGAAGGGGAACTGCCGGAATATAAACCATTAATGACCCGGTGAGTTTGTTTATTAGGTTAAAAGAAGGTGAAGACATGCAGCAATACGTCCTTGTTTCTTATGATATTTCGGACCAAAAAAGATGGAGAAAAGTATTTAAACTGATGAAAGGATACGGAGAACATGTTCAATATTCCGTATTCATATGCCAGTTAACTGAATTACAGAAGGCAAAATTACAAGCCTCTTTAGAAGACATTATCCATCATAAGAATGACCAAGTAATGTTTGTTCACATCGGGCCAGTGAAAGATGGTCAACTATCTAAAAAAATCTCAACAATTGGGAAAGAATTTGTTCCATTGGATTTAAAGCGGCTTATATTTTGAAAAGATATAGCAAAGAAATCTTATGAAAAAAATACAAAAATATATTGTTAAAAAATAGGGAATATTATATAATGGACTTACGAGGTTCTGTCTTTTGGTCAGGACAACCGTCTAGCTATAAGTGCTGCAGGGGTGTGAGAAACTCCTATTGCTGGACGATGTCTCTTTTATTTCTTTTTTCTTGGATCTGAGTACGAGCACCCACATTGGACATTTCGCATGGTGGGTGCTCGTACTATAGGTAAAACAAACCTTTTTAAGAAGAATACAAAAATAACCACAATATTTTTTAAAAGGAATTTTGATGGATTTACATAACCTCTCGCAACATGCTTCTAAAACCCAAGCCCACCATAGCCCAAAACCCCCTGCGGTCCAAGAAAAAAGAAATGATACGAGGCATTAGCACCGGGGAGAAGTCATTTAATAAGGCCACTGTTAAAAGTCCAAGAAAAAAGAAATGATACGAGGCATTAGCACAACAATATAAACGACTACTTTACCGTGTTCAAGAAAAAAGAAATGATATGAGGCATTAGCACGATGGGATGGGAGAGAGAGGACAGTTCTACTCTTGCTGTATCCAGCTTCTTTTACTTTATCCGGTATCATTTCTTCACTTCTTTCTGCACATAAAAAAGCACCTAACTATTTGGATAAGTTAAGTGCTTTTATTTCCGTTTGAAGTTGTCTATTGCTTTTTTCTTCATATCTTCAAATTTTTTCTGTTTCTCAGAGTCAACTTTACCAACTGTAATCCCTTTTCTTTTTGGCATTGGGGTATCTTTCCACCTTAGTGTGTTCATAAGGCTTATATTTATCACTCATTGTATTCCTCCAACACAATTATAATTTTTCCGTCATCCTCAATCCAACCGTCAACTGTGACAAAAGACGAATCTCTCTTAT >C-locusGTTTCATTTGGAAAGGGAGAGCATTGGCTTTTCTCTTTGTAAATAAAGTGCAAGCTTTGTAATAAGCTTCTAGTGGAGAAGTGATTGTTTGAATCACCCAATGCACACGCACTAAAGTTAGACGAACCTATAATTCGTATTAGTAAGTATAGTACATGAAGAAAAATGCAACAAGCATTTACTCTCTTTTAAATAAAGAATTGATAGCTGTTAATATTGATAGTATATTATACCTTATAGATGTTCGATTTTTTTTGAAATTCAAAAATCATACTTAGTAAAGAAAGGAAATAACGTCATGGACAAGCGAAAGCGTAGAAGTTACGAGTTTAGGTGGGAAGCGGGAGGCACCAGTCATGGCAATCCGTAGCATAAAACTAAAACTAAAAACCCACACAGGCCCGGAAGCGCAAAACCTCCGAAAAGGAATATGGCGGACGCATCGGTTGTTAAATGAAGGCGTCGCCTATTACATGAAAATGCTCCTGCTCTTTCGTCAGGAAAGCACTGGTGAACGGCCAAAAGAAGAACTACAGGAAGAACTGATTTGTCACATACGCGAACAGCAACAACGAAATCAGGCAGATAAAAATACGCAAGCGCTTCCGCTAGATAAGGCACTGGAAGCTTTGCGCCAACTATATGAACTGCTTGTCCCCTCCTCGGTCGGACAAAGTGGCGACGCCCAGATCATCAGCCGAAAGTTTCTCAGCCCGCTCGTCGATCCGAACAGCGAAGGCGGCAAAGGTACTTCGAAGGCAGGGGCAAAACCCACTTGGCAGAAGAAAAAAGAAGCGAACGACCCAACCTGGGAACAGGATTACGAAAAATGGAAAAAAAGACGCGAGGAAGACCCAACCGCTTCTGTGATTACTACTTTGGAGGAATACGGCATTAGACCGATCTTTCCCCTGTACACGAACACCGTAACAGATATCGCGTGGTTGCCACTTCAATCCAATCAGTTTGTGCGAACCTGGGACAGAGACATGCTTCAACAAGCGATTGAAAGACTGCTCAGTTGGGAGAGCTGGAACAAACGTGTCCAGGAAGAGTATGCCAAGCTGAAAGAAAAAATGGCTCAACTGAACGAGCAACTCGAAGGCGGTCAGGAATGGATCAGCTTGCTAGAGCAGTACGAAGAAAACCGAGAGCGAGAGCTTAGGGAAAACATGACCGCTGCCAATGACAAGTATCGGATTACCAAGCGGCAAATGAAAGGCTGGAACGAGCTGTACGAGCTATGGTCAACCTTTCCCGCCAGTGCCAGTCACGAGCAATACAAAGAGGCGCTCAAGCGTGTGCAGCAGCGACTGAGAGGGCGGTTTGGGGATGCTCATTTCTTCCAGTATCTGATGGAAGAGAAGAACCGCCTGATCTGGAAGGGGAATCCGCAGCGTATCCATTATTTTGTCGCGCGCAACGAACTGACGAAACGGCTGGAGGAAGCCAAGCAAAGCGCCACGATGACGTTGCCCAATGCCAGGAAGCATCCATTGTGGGTGCGCTTCGATGCACGGGGAGGAAATTTGCAAGACTACTACTTGACGGCTGAAGCGGACAAACCGAGAAGCAGACGTTTTGTAACGTTTAGTCAGTTGATATGGCCAAGCGAATCGGGATGGATGGAAAAGAAAGACGTCGAGGTCGAGCTAGCTTTGTCCAGGCAGTTTTACCAGCAGGTGAAGTTGCTGAAAAATGACAAAGGCAAGCAGAAAATCGAGTTCAAGGATAAAGGTTCGGGCTCGACGTTTAACGGACACTTGGGGGGAGCAAAGCTACAACTGGAGCGGGGCGATTTGGAGAAGGAAGAAAAAAACTTCGAGGACGGGGAAATCGGCAGCGTTTACCTTAACGTTGTCATTGATTTCGAACCTTTGCAAGAAGTGAAAAATGGCCGCGTGCAGGCGCCGTATGGACAAGTACTGCAACTCATTCGTCGCCCCAACGAGTTTCCCAAGGTCACTACCTATAAGTCGGAGCAACTTGTTGAATGGATAAAAGCTTCGCCACAACACTCGGCTGGGGTGGAGTCGCTGGCATCCGGTTTTCGTGTAATGAGCATAGACCTTGGGCTGCGCGCGGCTGCAGCGACTTCTATTTTTTCTGTAGAAGAGAGTAGCGATAAAAATGCGGCTGATTTTTCCTACTGGATTGAAGGAACGCCGCTGGTCGCTGTCCATCAGCGGAGCTATATGCTCAGGTTGCCTGGTGAACAGGTAGAAAAACAGGTGATGGAAAAACGGGACGAGCGGTTCCAGCTACACCAACGTGTGAAGTTTCAAATCAGAGTGCTCGCCCAAATCATGCGTATGGCAAATAAGCAGTATGGAGATCGCTGGGATGAACTCGACAGCCTGAAACAAGCGGTTGAGCAGAAAAAGTCGCCGCTCGATCAAACAGACCGGACATTTTGGGAGGGGATTGTCTGCGACTTAACAAAGGTTTTGCCTCGAAACGAAGCGGACTGGGAACAAGCGGTAGTGCAAATACACCGAAAAGCAGAGGAATACGTCGGAAAAGCCGTTCAGGCATGGCGCAAGCGCTTTGCTGCTGACGAGCGAAAAGGCATCGCAGGTCTGAGCATGTGGAACATAGAAGAATTGGAGGGCTTGCGCAAGCTGTTGATTTCCTGGAGCCGCAGGACGAGGAATCCGCAGGAGGTTAATCGCTTTGAGCGAGGCCATACCAGCCACCAGCGTCTGTTGACCCATATCCAAAACGTCAAAGAGGATCGCCTGAAGCAGTTAAGTCACGCCATTGTCATGACTGCCTTGGGGTATGTTTACGACGAGCGGAAACAAGAGTGGTGCGCCGAATACCCGGCTTGCCAGGTCATTCTGTTTGAAAATCTGAGCCAGTACCGTTCTAACCTGGATCGCTCGACCAAAGAAAACTCCACCTTGATGAAGTGGGCGCATCGCAGCATTCCGAAATACGTCCACATGCAGGCGGAGCCATACGGGATTCAGATTGGCGATGTCCGGGCGGAATATTCCTCTCGTTTTTACGCCAAGACAGGAACGCCAGGCATTCGTTGTAAAAAGGTGAGAGGCCAAGACCTGCAGGGCAGACGGTTTGAGAACTTGCAGAAGAGGTTAGTCAACGAGCAATTTTTGACGGAAGAACAAGTGAAACAGCTAAGGCCCGGCGACATTGTCCCGGATGATAGCGGAGAACTGTTCATGACCTTGACAGACGGAAGCGGAAGCAAGGAGGTCGTGTTTCTCCAGGCCGATATTAACGCGGCGCACAATCTGCAAAAACGTTTTTGGCAGCGATACAATGAACTGTTCAAGGTTAGCTGCCGCGTCATCGTCCGAGACGAGGAAGAGTATCTCGTTCCCAAGACAAAATCGGTGCAGGCAAAGCTGGGCAAAGGGCTTTTTGTGAAAAAATCGGATACAGCCTGGAAAGATGTATATGTGTGGGACAGCCAGGCAAAGCTTAAAGGTAAAACAACCTTTACAGAAGAGTCTGAGTCGCCCGAACAACTGGAAGACTTTCAGGAGATCATCGAGGAAGCAGAAGAGGCGAAAGGAACATACCGTACACTGTTCCGCGATCCTAGCGGAGTCTTTTTTCCCGAATCCGTATGGTATCCCCAAAAAGATTTTTGGGGCGAGGTGAAAAGGAAGCTGTACGGAAAATTGCGGGAACGGTTTTTGACAAAGGCTCGGTAAGGGTGTGCAAGGAGAGTGAATGGCTTGTCCTGGATACCTGTCCGCATGCTAAATGAAATTCAGTATTGTGAGCGACTGTACCATATTATGCATGTGCAGGGGCTGTTTGAGGAAAGCGCAGACACGGTCGAAGGAGCAGCACAACACAAGCGTGCAGAGACACATCTGCGCAAAAGCAAGGCAGCGCCGGAAGAGATGTGGGGGGACGCTCCGTTTAGCTTGCAGCTCGGCGACCCTGTGCTTGGCATTACGGGAAAGCTGGATGCCGTCTGTCTGGAAGAAGGTAAGCAGTGGATTCCGGTAGAAGGAAAGCATTCGGCGTCGCCAGAAGGCGGGCAGATGTTCACTGTAGGCGTGTATTCGCTGGACGGTTCTGCCTGGCCCAACGACCAAATCCAATTGTGTGCGCAAGGCTTGCTGCTTCGCGCGAATGGATATGAATCCGATTATGGCTACTTATACTACCGTGGCAATAAAAAGAAGGTTCGCATTCCTTTTTCGCAGGAACTCATAGCGGCTACTCACGCCTGCATTCAAAAAGCTCATCAGCTTCGGGAAGCCGAAATTCCCCCTCCGTTGCAGGAGTCGAAAAAGTGCTTTCGATGCTCGTTAAATTACGTATGCATGCCTGACGAGACGAATTACATGTTGGGGTTGAGCGCAAACATCAGAAAGATTGTGCCCAGTCGTCCAGATGGCGGGGTACTGTATGTTACAGAGCAGGGGGCAAAACTGGGCAGAAGCGGAGAAAGCTTGACCATCACCTGCCGGGGCGAAAAGATAGACGAAATCCCGATCAAAGACTTGATTCACGTGAGCTTGATGGGGCATGTGCAATGCTCTACGCAGCTTCTGCACACCTTGATGAACTGTGGCGTCCACGTCAGCTACTTGACTACGCATGGCACATTGACAGGAATAATGACTCCCCCTTTATCGAAAAACATTCGAACAAGAGCCAAGCAGTTTATCAAATTTCAGCACGCGGAGATCGCCCTTGGAATCGCGAGAAGGGTCGTGTATGCGAAAATTTCCAATCAGCGCACGATGCTGCGCCGCAATGGCTCACCAGATAAAGCAGTTTTAAAAGAGTTAAAAGAGCTTAGAGATCGCGCGTGGGAGGCGCCATCACTGGAAATAGTGAGAGGTATCGAGGGACGTGCAGCACAGTTGTACATGCAGTTTTTCCCTACCATGTTAAAGCACCCAGTAGTAGACGGTATGGCGATCATGAACGGTCGCAACCGTCGCCCGCCCAAAGATCCGGTCAATGCGCTGCTCTCCCTCGGCTATACGCTTCTTTCACGGGATGTTTACTCCGCATGTGCCAATGTCGGACTCGATCCACTGTTCGGCTTTTTCCATACGATGGAGCCGGGCAGACCAGCTTTGGCACTCGATCTGATGGAACCGTTCCGCGCCTTGATTGCCGATAGCGTAGCGATACGTACCTTGAATACGGAGGAACTCACCCTCGGGGACTTTTATTGGGGAAAAGACAGTTGTTATTTGAAAAAGGCAGGAAGACAAACGTATTTCGCTGCCTATGAAAGACGGATGAACGAGACGCTGACGCATCCGCAATTTGGGTATAAGCTCAGCTATCGCCGTATGCTGGAGCTGGAAGCAAGGTTTTTGGCCCGGTATCTGGATGGAGAGCTGGTGGAATATACGCCGCTCATGACAAGGTAGGAAATGACCATGCGACAATTTGTTCTGGTAAGCTATGATATTGCCGATCAAAAACGTTGGAGAAAAGTATTCAAGCTGATGAAGGGGCAAGGCGAGCACGTCCAGTACTCGGTGTTTCTGTGCCAACTCACCGAGATTCAGCAAGCCAAGCTAAAGGTAAGCCTGGCGGAGCTGGTTCACCATGGAGAAGACCAGGTCATGTTTGTAAAAATCGGCCCAGTGACGAGAGATCAACTGGACAAGCGGATATCTACTGTTGGCAGGGAGTTTCTGCCTCGCGATTTGACCAAATTTATCTATTAAGGAATGAAGAAAGCTAGTTGTAACAAAAGTGGAAAAAGAGTAAAATAAAGGTGTCAGTCGCACGCTATAGGCCATAAGTCGACTTACATATCCGTGCGTGTGCATTATGGGCCCATCCACAGGTCTATTCCCACGGATAATCACGACTTTCCACTAAGCTTTCGAATTTTATGATGCGAGCATCCTCTCAGGTCAAAAAAGCCGGGGGATGCTCGAACTCTTTGTGGGCGTAGGCTTTCCAGAGTTTTTTAGGGGAAGAGGCAGCCGATGGATAAGAGGAATGGCGATTGAATTTTGGCTTGCTCGAAAAACGGGTCTGTAAGGCTTGCGGCTGTAGGGGTTGAGTGGGAAGGAGTTCGAAAGCTTAGTGGAAAGCTTCGTGGTTAGCACCGGGGAGAAGTCATTTAATAAGGCCACTGTTAAAAGTTCGAAAGCTTAGTGGAAAGCTTCGTGGTTAGCACGCTAAAGTCCGTCTAAACTACTGAGATCTTAAATCGGCGCTCAAATAAAAAACCTCGCTAATGCGAGGTTTCAGC >D-locusGAAGTTATGTTGATAAAATGGTTTATGAAAACGTGAGTCTGTGGTAGTATTATAAACAATGATGGAATAAAGTGTTTTTTGCGCCGCACGGCATGAATTCAGGGGTTAGCTTGGTTTTGTGTATAAATAAATGTTCTACATATTTATTTTGTTTTTTGCGCCGCAAAATGCAACTGAAAGCCGCATCTAGAGCACCCTGTAGAAGACAGGGTTTTGAGAATAGCCCGACATAGAGGGCAATAGACACGGGGAGAAGTCATTTAATAAGGCCACTGTTAAAAGTTTTGAGAATAGCCCGACATAGAGGGCAATAGACTTTTGCTTCGTCACGGATGGACTTCACAATGGCAACAACGTTTTGAGAATAGCCCGACATAGTTATAGAGATGTATAAATATAACCGATAAACATTGACTAATTTGTTGAAGTCAGTGTTTATCGGTTTTTTGTGTAAATATAGGAGTTGTTAGAATGATACTTTTTGCCTAATTTTGGAACTTTATGAGGATATAAGATAGACTTGATAAAAAGGTAAAAGAAAGGTTAAAGAGCATGGCAGGAATAGTGACCTGTGATGAAGATGATGGTAGAATTAAAAGTGTTCTTAAAGAAAAACAATATTGGATAAGGAAAATAATTCAATAGATAAAAAATTTAGGGGGAAAAATGAAAATATCAAAAGTCGATCATACCAGAATGGCGGTTGCTAAAGGTAATCAACACAGGAGAGATGAGATTAGTGGGATTCTCTATAAGGATCCGACAAAGACAGGAAGTATAGATTTTGATGAACGATTCAAAAAACTGAATTGTTCGGCGAAGATACTTTATCATGTATTCAATGGAATTGCTGAGGGAAGCAATAAATACAAAAATATTGTTGATAAAGTAAATAACAATTTAGATAGGGTCTTATTTACAGGTAAGAGCTATGATCGAAAATCTATCATAGACATAGATACTGTTCTTAGAAATGTTGAGAAAATTAATGCATTTGATCGAATTTCAACAGAGGAAAGAGAACAAATAATTGACGATTTGTTAGAAATACAATTGAGGAAGGGGTTAAGGAAAGGAAAAGCTGGATTAAGAGAGGTATTACTAATTGGTGCTGGTGTAATAGTTAGAACCGATAAGAAGCAGGAAATAGCTGATTTTCTGGAGATTTTAGATGAAGATTTCAATAAGACGAATCAGGCTAAGAACATAAAATTGTCTATTGAGAATCAGGGGTTGGTGGTCTCGCCTGTATCAAGGGGAGAGGAACGGATTTTTGATGTCAGTGGCGCACAAAAGGGAAAAAGCAGCAAAAAAGCGCAGGAGAAAGAGGCACTATCTGCATTTCTGTTAGATTATGCTGATCTTGATAAGAATGTCAGGTTTGAGTATTTACGTAAAATTAGAAGACTGATAAATCTATATTTCTATGTCAAAAATGATGATGTTATGTCTTTAACTGAAATTCCGGCAGAAGTGAATCTGGAAAAAGATTTTGATATCTGGAGAGATCACGAACAAAGAAAGGAAGAGAATGGAGATTTTGTTGGATGTCCGGACATACTTTTGGCAGATCGTGATGTGAAGAAAAGTAACAGTAAGCAGGTAAAAATTGCAGAGAGGCAATTAAGGGAGTCAATACGTGAAAAAAATATAAAACGATATAGATTTAGCATAAAAACGATTGAAAAGGATGATGGAACATACTTTTTTGCAAATAAGCAGATAAGTGTATTTTGGATTCATCGCATTGAAAATGCTGTAGAACGTATATTAGGATCTATTAATGATAAAAAACTGTATAGATTACGTTTAGGATATCTAGGAGAAAAAGTATGGAAGGACATACTCAATTTTCTCAGCATAAAATACATTGCAGTAGGCAAGGCAGTATTCAATTTTGCAATGGATGATCTGCAGGAGAAGGATAGAGATATAGAACCCGGCAAGATATCAGAAAATGCAGTAAATGGATTGACTTCGTTTGATTATGAGCAAATAAAGGCAGATGAGATGCTGCAGAGAGAAGTTGCTGTTAATGTAGCATTCGCAGCAAATAATCTTGCTAGAGTAACTGTAGATATTCCGCAAAATGGAGAAAAAGAGGATATCCTTCTTTGGAATAAAAGTGACATAAAAAAATACAAAAAGAATTCAAAGAAAGGTATTCTGAAATCTATACTTCAGTTTTTTGGTGGTGCTTCAACTTGGAATATGAAAATGTTTGAGATTGCATATCATGATCAGCCAGGTGATTACGAAGAAAACTACCTATATGACATTATTCAGATCATTTACTCGCTCAGAAATAAGAGCTTTCATTTCAAGACATATGATCATGGGGATAAGAATTGGAATAGAGAACTGATAGGAAAGATGATTGAGCATGATGCTGAAAGAGTCATTTCTGTTGAGAGGGAAAAGTTTCATTCCAATAACCTGCCGATGTTTTATAAAGACGCTGATCTAAAGAAAATATTGGATCTCTTGTATAGCGATTATGCAGGACGTGCATCTCAGGTTCCGGCATTTAACACTGTCTTGGTTCGAAAGAACTTTCCGGAATTTCTTAGGAAAGATATGGGCTACAAGGTTCATTTTAACAATCCTGAAGTAGAGAATCAGTGGCACAGTGCGGTGTATTACCTATATAAAGAGATTTATTACAATCTATTTTTGAGAGATAAAGAGGTAAAGAATCTTTTTTATACTTCATTAAAAAATATAAGAAGTGAAGTTTCGGACAAAAAACAAAAGTTAGCTTCAGATGATTTTGCATCCAGGTGTGAAGAAATAGAGGATAGAAGTCTTCCGGAAATTTGTCAGATAATAATGACAGAATACAATGCGCAGAACTTTGGTAATAGAAAAGTTAAATCTCAGCGTGTTATTGAAAAAAATAAGGATATTTTCAGACATTATAAAATGCTTTTGATAAAGACTTTAGCAGGTGCTTTTTCTCTTTATTTGAAGCAGGAAAGATTTGCATTTATTGGTAAGGCAACACCTATACCATACGAAACAACCGATGTTAAGAATTTTTTGCCTGAATGGAAATCCGGAATGTATGCATCGTTTGTAGAGGAGATAAAGAATAATCTTGATCTTCAAGAATGGTATATCGTCGGACGATTCCTTAATGGGAGGATGCTCAATCAATTGGCAGGAAGCCTGCGGTCATACATACAGTATGCGGAAGATATAGAACGTCGTGCTGCAGAAAATAGGAATAAGCTTTTCTCCAAGCCTGATGAAAAGATTGAAGCATGTAAAAAAGCGGTCAGAGTGCTTGATTTGTGTATAAAAATTTCAACTAGAATATCTGCGGAATTTACTGACTATTTTGATAGTGAAGATGATTATGCAGATTATCTTGAAAAATATCTCAAGTATCAGGATGATGCCATTAAGGAATTGTCAGGATCTTCGTATGCTGCGTTGGATCATTTTTGCAACAAGGATGATCTGAAATTTGATATCTATGTAAATGCCGGACAGAAGCCTATCTTACAGAGAAATATCGTGATGGCAAAGCTTTTTGGACCAGATAACATTTTGTCTGAAGTTATGGAAAAGGTAACAGAAAGTGCCATACGAGAATACTATGACTATCTGAAGAAAGTTTCAGGATATCGGGTAAGGGGAAAATGTAGTACAGAGAAAGAACAGGAAGATCTGCTAAAGTTCCAAAGATTGAAAAACGCAGTAGAATTCCGGGATGTTACTGAATATGCTGAGGTTATTAATGAGCTTTTAGGACAGTTGATAAGTTGGTCATATCTTAGGGAGAGGGATCTATTATATTTCCAGCTGGGATTCCATTACATGTGTCTGAAAAACAAATCTTTCAAACCGGCAGAATATGTGGATATTCGTAGAAATAATGGTACGATTATACATAATGCGATACTTTACCAGATTGTTTCGATGTATATTAATGGACTGGATTTCTATAGTTGTGATAAAGAAGGGAAAACGCTCAAACCAATTGAAACAGGAAAGGGCGTAGGAAGTAAGATAGGACAATTTATAAAGTATTCCCAGTATTTATACAATGATCCGTCATATAAGCTTGAGATCTATAATGCAGGATTAGAAGTTTTTGAAAACATTGATGAACATGATAATATTACAGATCTTAGAAAGTATGTGGATCATTTTAAGTATTATGCATATGGTAATAAAATGAGCCTGCTTGATCTGTATAGTGAATTCTTCGATCGTTTCTTTACATATGATATGAAGTATCAGAAGAATGTAGTGAATGTGTTGGAGAATATCCTTTTAAGGCATTTTGTAATTTTCTATCCGAAGTTTGGATCAGGAAAAAAAGATGTTGGAATTAGGGATTGTAAAAAAGAAAGAGCTCAGATTGAAATAAGTGAGCAGAGCCTCACATCGGAAGACTTCATGTTTAAGCTTGACGACAAAGCAGGAGAAGAAGCAAAGAAGTTTCCGGCAAGGGATGAACGTTATCTCCAGACAATAGCCAAGTTGCTCTATTATCCTAACGAAATTGAGGATATGAACAGATTCATGAAGAAAGGAGAAACGATAAATAAAAAAGTTCAGTTTAATAGAAAAAAGAAGATAACCAGGAAACAAAAGAATAATTCATCAAACGAGGTATTGTCTTCAACTATGGGTTATTTATTTAAGAACATTAAATTGTAAAAAAGATTCGTTGTAGATAATTGATAGGTAAAAGCTGACCGGAGCCTTTGGCTCCGGACAGTTGTATATAAGAGGATATTAATGACTGAAAATGATTTTTGTTGGAAGTCAGTTTTTTCTGTGGAAAGCGAAATCGAATATGATGAGTATGCATATGGCAGAAGAGCTGTAGAAGGCGAGAATACATATGATTACATTACTAAGGAAGAAAGACCGGAACTTAATGACGAATATGTAGCGAGACGTTGCATTTTCGGTAAAAAAGCAGGAAAAATATCCAGGTCGGATTTTAGTAGGATAAGATCTGCGTTGGATCATGCGATGATAAATAATACACATACAGCATTTGCCAGATTTATCACTGAAAATCTGACGAGACTCAATCACAAAGAACATTTTCTGAATGTGACACGTGCATATTCTAAACCTGATTCTGAAAAATTGATACAACCGAGATACTGGCAGTCGCCTGTAGTTCCAAAGGATAAACAAATATATTATAGCAAGAATGCGATTAAAAAATGGTGTGGTTACGAAGATGATATTCCGCCTCGTTCTGTGATAGTTCAGATGTGTCTATTGTGGGGGACTGATCATGAAGAGGCAGATCATATCCTTCGCAGTTCAGGATACGCGGCGCTTAGTCCTGTTGTACTTCGAGATCTTATCTATATGTATTATCTGGATCATCAGGATTTGCAAAAAAATGAGTTGATATGGGAAGTAAAAAAGCAGTTGGATCACTTCGATTTGACAAATAGAAATTATGATACAAATCCTTTTGATGTAGGGGGCAGCGTAAATGATCATATCTGTGAACTGAGCGAGCATATAGCGAAGGCTCATTATATTTATGAGAGGGCTAAGGAAGGACCATTGCAAAATGTAATTCGGGATATTTTGGGAGATACACCTGCCCTTTATTCTGAAATGGCATTTCCTCAGCTAGCATCTATAAACAGGTGTGCTTGCAATTCGCTTTCTTCATATCAAAAAAATATTTTTGATACTGACATAGCTATATATGCAGATGAAAAGGACACAAGAGGTAAATCAGACCGTATCCTTGTTGAGGGCGCATCTTCGAAATGGTATGAATTGAAGAAACGCGATGCTAATAATGTCAAAATTTCTGAAAAGCTGAGTATACTCAATACTATTCTTAAATTTAATAGTGTTTTTTGGGAAGAATGTTACCTTGATGGAAATATAAAACAATCGAGCGGAAAGCGATCTGAGGCAGGAAAAATTCTTTATGGTCGCGACAACGGAAAAGAAAATGTCGGAGTTTCAAAATTGGAATTGGTGCGGTATATGATAGCTGCAGGTCAGGAACAAAATCTGGGAAATTACCTGGTGAGTTCAGGATTTTGGAGAAAAAATCATATGCTGTCATTTATACAAGGCAATGATATAGCGCTTGATGAGATGGATGAATTGGATCTCTTAGACTATATTCTGATATATGCATGGGGATTTAGGGAAAATATCATTAAAAAGAACAGTAATGTGAATTCTTTGGATGAAAAGACTAGAAAAGTGCAGTTTCCGTTTATAAAGTTACTCATGGCAATTGCAAGAGATATCCAGATACTTATATGTTCAGCACATGAAAAAACAGTCGATGAGTCATCTCGAAATGCAGCAAAGAAGATAGATATATTGGGAAATTATATTCCTTTTCAGATTCATCTTCAGAGAACTAAAAAAGATGGTGGAAGAGTGGTAATGGATACATTGTGTGCTGATTGGATTGCGGATTATGAATGGTACATTGATCTTGAGAAAGGAACACTTGGATGAGCAGTGATGAAAGGATATTTAAAAAATTTTTGGAAAAAGGATCGATTTCTGAGCAGAAAAAGATGCTTTTAGAAGAAAAGAAATGTTCGGATAAACTAACTGCACTGCTTGGGAATTACTGCATACCGATAGACAATATTTCAGAGTCAGACGGAAAAATATATGCGGTCTATAAGCTTCCAAAAAATGTTAAACCTTTGTCCGAAATCATTAATGATGTATCCTTTTCTGATTGTACGATGAGAGTACGTTTGCTTCTCATAAAGAGAATTCTGGAACTCGTGTGTGCTTTTCACGAAAAAAAATGGTATTGTCTCAGTATTTCACCGGGAATGCTCATGGTTGAAGATTTTGATATACCGATGGGAAATGTCGGAAAAGTATTGATATATGATTTCAGAAATCCTGTTCCGTTCGAGTCAGTAAATGAAAGACATAATTTTAACGTTTCAAATAAATACACTTCACCGGAGCTGCTCATCCATTCAAGATATGACGAGTCGAAATCTGTGAGTGAAAAATCAGATTTGTATTCTGTTGCAAAAATTGCGGAAACAATAATAGGAGATTTTAACAGTATTATTGCAAATGGAAATTTGATACTACTTGCAATGCTTAGAGTTTTTATCAGTACAGGGAAAAGTCCGGAACCTGAGTATCGGTTTGAATCGTCGGAAAATATGCTTTCAGTATTTGAAAATTTGATCAAAGAAAATTGTTTTTTTGAAAAAAACGATTATACATCTATGTTTCATCAGGCGTATGACAATTTTTTTGAATGGCAGGAATGTTTGATATCACCGGATCACTTGGATAAAAATATGTTCGAGGCAGCTTTATCAAATCTTGAGGATCAGCTGCTTAGGGTTGATATTGATAAGTATAGAGCAGAGTACTTCTATAAGCTTCTCCGAGAGTTGTCTAATAAATATAAAAATACAATTACTGATGAACAAAAGGTAAGGTTGGCAATACTTGGAATCAGAGCGAAAAATAATCTGGGAAAAAGTTTTGATGCATTGGAAATATATGAGTCAGTACGTGATTTAGAAACTATGTTGGAGGAGATGGCAGAGCTTAGTCCTGTCATTGCTTCGACATATATGGATTGCTACCGATATGCAGATGCGCAGAAAGTGGCGGAAGAAAACATTATCAGGCTTCATAATAGTAATATTCGTATGGAGAAAAAAAGAATACTGCTTGGAAGGTCATATAGTTCAAAAGGGTGCAGCATGGGGTTTCAGCATATTCTTGGTGCGGATGAGTCATTTGAACAGGCTTTATATTTCTTTAACGAAAAGGACAATTTTTGGAAAGAAATATTTGAGAGCAGAAATTTAGAGGACAGCGATAGACTTATAAAGTCTTTACGAAGCAATACGCATATTACGCTGTTTCATTACATGCAATATGCATGTGAAACAAGGAGAAAGGAATTATATGGAGCACTTTCAGACAAATATTTTATAGGTAAAGAATGGACAGAAAGACTCAAAGCATATATAAGCAACAAGGATATATGGAAAAACTATTATGAGATATATATTCTGCTAAAGGGTATTTATTGCTTCTATCCAGAAGTCATGTGTTCGTCTGCGTTTTATGATGAAATCCAAAAAATGTACGATCTTGAATTTGAAAAGGAAAAAATGTTTTACCCATTGAGTCTGATAGAACTGTATCTTGCTCTGATAGAGATAAAAGTTAATGGGAGTCTGACGGAGAATGCCGAGAAGTTGTTTAAACAGGCATTGACACATGACAATGAAGTCAAAAAAGGAAATATGAATATTCAGACCGCCATTTGGTATCGAATATATGCACTGTATAACGATGTAAAAGATGAAACTGATAAGAATAAAAGGCTTTTAAAACGGCTTATGATTCTTTGCCGACGATTTGGTTGGGCGGATATGTATAGTGCTTTGGAGAAGGATGGGAAGTTAATTGATTTTTTGAGATTTGAGGTATGTTAAATGATAACACTTGCATTAGATGAAAATGGCAAATTTGAAGATGCTTTTTCTAAAAAAAATGAAAAACCGATAATGATTGCGGGGATAATCTATGATGACAAGGGGAAAGAGTATGATGCTGAGAATGAACGCTACAGGATATCCAGTTATCTGCGAGCAGTATGTGACAGTTTGGGTGCGAAATACCCTCAGGATCTACATTCAAATAGTAATGGAAATAAGGCGACTGTTGGGAAAGTAAAATGTAAAATTGGTGAAACACTAAAGGAATTCTTGAGAGAAGGAACCTATGAAAAAAAGGAATTGCCGACAAAGAACGGTTATTTAAATAAGAGATCTGGAAAATATGTAATGTTTGCAGAACTCAGGAGTAGTCAGGGAGTTAAAAAGCGTGTTAGTGGTTGGAATGACAATGATCTGACTCAGGATGAAAAGGTCAGCAATCTGTACCTTCATATGGCAGAAAATGCCGTTGTCAGAATGCTCTTCCATAATCCTATATATGAAGATGTAACAGATGTAAATCTCTATTTTCCCACGCGAAAAGTTGTTCTGAAAGATAGAGATAGAGAATACGATAAACAAGATTTCAAAATATATGGTGATAAGGACAAGTGCGAAGCAGAAAGCGGGAGATTGGTGCATTATGATATCGTGTCATCGGATTTTTACCGTACGATAATGGAGAACGAATGTACAAGAATTAATAAAAAGCAATTAAATGTTCATTATATGAACACAAGCCCAATTTCGTACTGGGAGAAAAATGAAAAATATAATACATTTTTATATTTGGCTGACATAGTTTGTTCTATGCTGGATTATTACAAAAAGGGTTCGAGTCCGGCAGAGTGGATGGATTCTTTTGCCGAATGGGGAAACAAATATTTTGGTGATGATCAGATAATCTTATTTGGGTATGATGATATAGATGACAAATACATGGAGGCTGTAGATGCAGTAGGACAGGGAGAGTATTTTCATGCGCTGGATATTATATATGATGCGGAATGTAGTGGAAGTGAATTTGAGAAGCACTACAAAGATTATTGGTTTCCAAAGCTTATAAAAAAGATACGAATAACAGCAACTGTGGATAATTTATGCAGATCGATCTCAGATCTGGAGAGTTTTACATATCGAAGTAATCTTGATCAGCAGAAACTTTTGTGGATTTTTGAGGAAATCAAAGCTATCGTCGATAAGGGAGATTTTGGAAAGAAATATCATACAGATCAGGTTATGTTTGATATGTGTAATGCCGGTATTGCTGTGTACAATCATATCGGAGATTTTGGGACTGCAAAGGAATACTATGATGAGTGCATGAAACACACTGGGGATGTGGATCTGGTAAAGATACTTCGTGCATCAAATAAAATGGTGGTCTTTCTTGACGATGCTTTTAGGTATGGTGACGCGACAGAACGTGCCAGGAAGAATGTTGAATACCAAAAAGCTTTGCACGATATAAAGAGTGAGATTTGTCCGGAAAAGAAAGATGAAGACTTGAACTATGCCATATCGCTCAGTCAATTTGGACAGGCGCTTGCGTGTGAAAAAAATTCTGATGCAGAGAGTGTTTTCCTAGAGTCGTTGCGGCATATGAGGAAAGGGACTGCCAATTATCAGATTACTCTTTCATATTTACTCCATTTTTATCTGGATATGGGAATGACAGATTCTTATCGAGAAAAAACAAAGGACTATTTTGGAAGTGAAAAACCAAAGGAACAGCTGAAAGAATTGCTGAAGTTATCGGGAAAGGATGATAGTATAGTTACTTTCAAATTTGCAATGTATGTCTATTTACGTGCACTTTGGGTATTACAGGAACCGCTTACTGATTTTATCAGAACAAGATTAGAGGACATACGTGAGACTCTTGTAAAGAAGAAAATGAGTGAACATATGGTTGGACATCCGTGGGAGTTGATTTATAAATATCTGGCATTTCTTTTTTATCGTGATGGAAATTGTGAAGCTGCTGAAAAATATATTCATAAAAGTGAAGAGTGCTTGGAAACACAAGGACTGACTATAGATGCGATTATTCATAATGGTAAGTATGAATATGCAGAATTGTCAGGTGACGAGGAGATGATGGCAAGAGAGAAAGCGTACTTTGATGAAAAAGGGATAGATAGAAAAAATGTTTGTACTTTTATGTATCATTGATGTTTAATAAGATTTGACCGAGGAGTGACAGGTAATCGCCGGTATATCTGGTATTACCTGTCATTTTTTGATGAAATAAGCTACTTTTTGCCTAAAAAACGAAACTGTTGGTGTTTTATGATGATTGTGTCAACAAAAGAGAGCAAAAGAAGAGGAGAAAAGTAATGTCAATGATTTCATGTCCGAATTGTGGTGGAGAGATATCTGAAAGGTCAAAGAAATGTGTTCATTGTGGATATGTGTTAGTCGAAGAAGCTAAAGTAGTGTGCACAGAATGTGGAACTGAGGTAGAGAGTGGCGCTGCTGTATGTCCGAAGTGCGGCTGTCCTGTAAATGATAGTGAGACGCCTCAGAAAGTTGAAGTGACTAGGGTAAATGTATCTTCCGTAATCAGCAAAAAAGTCGTTGTAAGCATACTGATCGCAGTGATTACAATTGCAGGTTTTTTCTATGGAGTGAAGTATTCGCAGGAAAAGAAAGCAATTGAAGAGTCAGTAAAGCAGAAGGAAGACTATCAAAGTACGCTAGAGCTTGCTTCGCTAATGATGCTTCAAGGAGCTTCGGATGCAGAAACTTGTGGGAATTTGGTTAGGAAAGTGTGGAGCAACTGCATTTATAAGGAGAGGGATGAAGAAACCGACAAGTATACGTGTGATAGCAGGGGTGCAGGATGGTTTTATGATGATTTTAATGATGCATTAATGGCTCTTTACAGTGACAGCAGTTTTGGCAAGAAGATAAATGAAATCAAAAACGGTCAGGAAACCGTTGCGGCGATGATGAAAGATCTGAAAAATCCGCCGGATGAGATGGCAGATGCCTATGAGGATATTCAAAATTTTTATGTGTCCTATCTAACGCTGACAGAAATGGTTGTGAATCCAACTGGAAGTTTGAGTTCTTTTTCATCTGATTTTTCCGATGCGGATACGGAGGTGTCCAATGCCTATAGCCGGATGAAGTTGTATTTAGATTAAACTATTGAGGAAAAAATGGAGGTGCTTTAATGCGGGGGAGAAACTGTGGAGGGTCATCAGGCGACGGACTGCTGGTACTTCTCGTACTGCTTGTCCTTTTTTATAAAATCATGCCATTCATAGGTTTATGGATTTTAATTTTTGGTGATGCTGAACGTAAAGATCTGGGTATGGGTATGATTATTGTCGGGATAGTTCTATATGTATTATTAGAGGTTTTTTAATGTGAGTTTCTGTGGTAAACTATAAAAGTACAAGCTTTTGCGCCGCACCGCATAAATAGCGGATTTATGACCATTATTTGGTGAAAAAAATGGTGTACACCTGTGTTTTTTTGTTTTGCGCCGCAAAATGCGCCACGGAACCGCATGCAGAGCACCCTGCAAGAGACAGGGTTATGAAAACAGCCCGACATAGAGGGCAATAGACACGGGGAGAAGTCATTTAATAAGGCCACTGTTAAAAGTTATGAAAACAGCCCGACATAGAGGGCAATAGACATAAAGACCAAAAACAGGTCATCTGCATACTGTGTTATGAAAACAGCCCGATATAGAGGGTGTGAGAGATATAGTTCTCGTCACAGTGCAGAAAATGACCTATTATGTGCCGAAAAACAAAATGAAAAAAGAATGGAAAGGCGTATTTAATGAAATGCTGATCTGTTGATTTGAATTAACAAAAAAAGGTCGCCCCACGGATGACAAAAACATCCGGGGGCGACCCTTTT >E-locusTACTGTGTGCATAAGTCTTCCTTAGATCCATAGGTACAGCAGTTTTATTTATTAGCCTTAGAAAATGGAAAATAGAGCTTATAAATGATATGATATTTATGAATAAAATGATTGCATTCTCGTGCAAACTTTAAATATATTGATTATATCCTTTACATTGGTTGTTTTAATTACTATTATTAAGTAGGAATACGATATACCTCTAAATGAAAGAGGACTAAAACCCGCCAAAAGTATCAGAAAATGTTATTGCAGTAAGAGACTACCTCTATATGAAAGAGGACTAAAACTTTTAACAGTGGCCTTATTAAATGACTTCTGTAAGAGACTACCTCTATATGAAAGAGGACTAAAACGTCTAATGTGGATAAGTATAAAAACGCTTATCCATCATTTAGGTGTTTTATTTTTTTGTGATTATATGTACAATAGAAGAGAGAAAAAAATCATTGAGGTGAAAACTATGAGAATTACTAAAGTAGAGGTTGATAGAAAAAAAGTACTAATTTCTAGGGATAAAAACGGGGGCAAGTTAGTTTATGAAAATGAAATGCAAGATAATACAGAACAAATCATGCATCACAAAAAAAGTTCTTTTTACAAAAGTGTGGTAAACAAAACTATTTGTCGTCCTGAACAAAAACAAATGAAAAAATTAGTTCATGGATTATTACAAGAAAATAGTCAAGAAAAAATAAAAGTTTCAGATGTCACTAAACTTAATATCTCAAATTTCTTAAATCATCGTTTCAAAAAAAGTTTATATTATTTTCCTGAAAATAGTCCTGACAAAAGCGAAGAATACAGAATAGAAATAAATCTCTCCCAATTGTTAGAAGATAGCTTAAAAAAACAGCAAGGGACATTTATATGTTGGGAATCTTTTAGCAAAGACATGGAATTATACATTAATTGGGCGGAAAATTATATTTCATCAAAAACGAAGCTAATAAAAAAATCCATTCGAAACAATAGAATTCAATCTACTGAATCAAGAAGTGGACAACTAATGGATAGATATATGAAAGACATTTTAAATAAAAACAAACCTTTCGATATCCAATCAGTTAGCGAAAAGTACCAACTTGAAAAATTGACTAGTGCTTTAAAAGCTACTTTTAAAGAAGCGAAGAAAAACGACAAAGAGATTAACTATAAGCTTAAGTCCACTCTCCAAAACCATGAAAGACAAATAATAGAAGAATTGAAGGAAAATTCCGAACTGAACCAATTTAATATAGAAATAAGAAAACATCTTGAAACTTATTTTCCTATTAAGAAAACAAACAGAAAAGTTGGAGATATAAGGAATTTAGAAATAGGAGAAATCCAAAAAATAGTAAATCATCGGTTGAAAAATAAAATAGTTCAACGCATTCTCCAAGAAGGGAAATTAGCTTCTTATGAGATTGAATCAACAGTTAACTCTAATTCCTTACAAAAAATTAAAATTGAAGAAGCATTTGCCTTAAAGTTTATCAATGCTTGTTTATTTGCTTCTAACAATTTAAGGAATATGGTATATCCTGTTTGCAAAAAGGATATATTAATGATAGGTGAATTTAAAAATAGTTTTAAAGAAATAAAACACAAAAAATTCATTCGTCAATGGTCGCAATTCTTCTCTCAAGAAATAACTGTTGATGACATTGAATTAGCTTCATGGGGGCTGAGAGGAGCCATTGCACCAATAAGAAATGAAATAATTCATTTAAAGAAGCATAGCTGGAAAAAATTTTTTAATAACCCTACTTTCAAAGTGAAAAAAAGTAAAATAATAAATGGGAAAACGAAAGATGTTACATCTGAATTCCTTTATAAAGAAACTTTATTTAAGGATTATTTCTATAGTGAGTTAGATTCTGTTCCAGAATTGATTATTAATAAAATGGAAAGTAGCAAAATTTTAGATTATTATTCCAGTGACCAGCTTAACCAAGTTTTTACAATTCCGAATTTCGAATTATCTTTACTGACTTCGGCCGTTCCCTTTGCACCTAGCTTTAAACGAGTTTATTTGAAAGGCTTTGATTATCAGAATCAAGATGAAGCACAACCGGATTATAATCTTAAATTAAATATCTATAACGAAAAAGCCTTTAATTCGGAGGCATTTCAGGCGCAATATTCATTATTTAAAATGGTTTATTATCAAGTCTTTTTACCGCAATTCACTACAAATAACGATTTATTTAAGTCAAGTGTGGATTTTATTTTAACATTAAACAAAGAACGGAAAGGTTACGCCAAAGCATTTCAAGATATTCGAAAGATGAATAAAGATGAAAAGCCCTCAGAATATATGAGTTACATTCAGAGTCAATTAATGCTCTATCAAAAAAAGCAAGAAGAAAAAGAGAAAATTAATCATTTTGAAAAATTTATAAATCAAGTGTTTATTAAAGGTTTCAATTCTTTTATAGAAAAGAATAGATTAACCTATATTTGCCATCCAACCAAAAACACAGTGCCAGAAAATGATAATATAGAAATACCTTTCCACACGGATATGGATGATTCCAATATTGCATTTTGGCTTATGTGTAAATTATTAGATGCTAAACAACTTAGCGAATTACGTAATGAAATGATAAAATTCAGTTGTTCCTTACAATCAACTGAAGAAATAAGCACATTTACCAAGGCGCGAGAAGTGATTGGTTTAGCTCTTTTAAATGGCGAAAAAGGATGTAATGATTGGAAAGAACTTTTTGATGATAAAGAAGCTTGGAAAAAGAACATGTCCTTATATGTTTCCGAGGAATTGCTTCAATCATTGCCGTACACACAAGAAGATGGTCAAACACCTGTAATTAATCGAAGTATCGATTTAGTAAAAAAATACGGTACAGAAACAATACTAGAGAAATTATTTTCCTCCTCAGATGATTATAAAGTTTCAGCTAAAGATATCGCAAAATTACATGAATATGATGTAACGGAGAAAATAGCACAGCAAGAGAGTCTACATAAGCAATGGATAGAAAAGCCCGGTTTAGCCCGTGACTCAGCATGGACAAAAAAATACCAAAATGTGATTAATGATATTAGTAATTACCAATGGGCTAAGACAAAGGTCGAATTAACACAAGTAAGGCATCTTCATCAATTAACTATTGATTTGCTTTCAAGGTTAGCAGGATATATGTCTATCGCTGACCGTGATTTCCAGTTTTCTAGTAATTATATTTTAGAAAGAGAGAACTCTGAGTATAGAGTTACAAGTTGGATATTATTAAGTGAAAATAAAAATAAAAATAAATATAACGACTACGAATTGTATAATCTAAAAAATGCCTCTATAAAAGTATCATCAAAAAATGATCCCCAGTTAAAAGTTGATCTTAAGCAATTACGATTAACCTTAGAGTACTTAGAACTTTTTGATAACCGATTGAAAGAAAAACGAAATAACATTTCACATTTTAATTACCTTAACGGACAGTTAGGGAACTCTATTTTAGAATTATTTGACGATGCTCGAGATGTACTTTCCTATGATCGTAAACTAAAGAATGCGGTGTCTAAATCTTTGAAAGAAATTTTAAGCTCTCATGGAATGGAAGTGACATTTAAACCACTATATCAAACCAATCATCATTTAAAAATTGATAAACTCCAACCTAAAAAAATACACCACTTAGGTGAAAAAAGTACTGTTTCTTCAAATCAAGTTTCTAATGAATACTGTCAACTAGTAAGAACGCTATTAACGATGAAGTAATTCTTTTAAAGCACATTAATTACCTCTAAATGAAAAGAGGACTAAAACTGAAAGAGGACTAAAACACCAGATGTGGATAACTATATTAGTGGCTATTAAAAATTCGTCGATATTAGAGAGGAAACTTTAGATGAAGATGAAATGGAAATTAAAAGAAAATGACGTTCGCAAAGGGGTGGTGGTCATTGAGTAAAATTGACATCGGAGAAGTAACCCACTTTTTACAAGGTCTAAAGAAAAGTAACGAAAACGCCCGAAAAATGATAGAAGACATTCAATCGGCTGTCAAAGCCTACGCTGATGATACAACTTTAAAAGGAAAAGCAGTGGATTCTTCACAAAGATACTTTGATGAAACGTATACTGTTATTTGTAAAAGTATCATAGAAGCATTAGATGAAAGCGAAGAGAGATTACAACAATATATTCATGATTTTGGAGATCAAGTGGATTCTTCACCTAACGCACGAATTGATGCGGAATTACTACAAGAAGCAATGAGTAGGTTAGCTGACATAAAGCGGAAGCAAGAAGCACTTATGCAATCCTTATCTTCTTCTACAGCAACGCTTTACGAAGGCAAGCAACAAGCGTTACACACTCAATTCACGGATGCGCTGGAGCAAGAAAAAATATTGGAACGCTATATTACTTTTGAACAAACTCACGGGAATTTTTTTGACTCATTTGGAGAACTTGTCTATCGAACGGGACAAGCAGTGCGTGAATTAGCTAATAACGTCACATTCGAGAGCCAAACAGGAAGCTATCATTTTGATAAAATAGATGCTTCTAGATTCCAAACTTTGCAAGAAATGTTGCCAAAGGCAAAGAAAAAAGCATTTAATTTTAATGACTACCAAATAACATGGAATGGCACCACGCACCTTTTATGGAAAAATGGTAAAGTGGATGCAGAAGCAACCAAAGCTTATAACGAGGCGAAACTGAATGGAAAGCTACCAAAGGAAGGTAATGTAGCAACACAAGATGCAGAACTATTAAAAGGCATTTTGGCTTCACTGAAAAACAAGAAAGATCCTATCACTGGAGCAGATATAAGCAGTGTGCATGTATTATCTATCCTTAGCGGGCTCGCATTCTCCTATACAGCTGGGAATTATAAGGGAAGAAAACTTACTGTTCCAAAAAGTTTCTTAGACAAATTAAAGAAAAACCGAAAATCTAAAGTACCTAAACTATCTAGTTTATCAGAAAAACAACAACTAAAACTCGCAAATAAATACAAGAAAAAATCACCTATTCCAATTCCAGATGATGCTAAAATCAAAGCTCAGACGAAAAAGGCTGGTTATGAACAAATATCTTATAAATGGAAAGAGAATGGGATAACCTTTGAAGTTAGATGGCATACTAGGACACCAGGTGCACCAAAGGAACAAGGAAATACGTTTGTTATAGAAAGAAAAATTCAGGGTACAGCAGAAGGGAAAACAAAAGTTCAACAAATATTGGTTGGAGATAATAAGTGGGTGAGTAAAAGTGAGTGGCAAAAGGCTATAACTGATAAGAAAAATGGTGTAAGTACCTCGGAGCAAAATAAAATGTTGTCTGATGGACATTGGAAAGAATAGAAAGGAGCAAAATGATGGAAGATTATTATAAAGGTTTTGAGGGATATCCAGAGATAGATTTTTATACGTATATAGATGATATGAAATTGGGTATAGCAATGTGGGAAGGATACTTTGACAACATTATGAAAGAAATTAATCCAAGTAACGGAAGATGGACTTCATTAGCGTATTATTATCATTTAGATGAGGGGTGGTATGATGAAAGTCCTTGGGAAATACCAAGTAATACAGAAGCATTAGAATTATTGGAAACAATCCATATATCTAATCTAGATACTATCACACAAGAGATATTACTTAAATTAATAAATTTATTAAAGAAGAATATAAATAGACAAGTTTATATTGAATACTCATAAAAAAGATGATTATGATATATTATAGAACAAACGAACAAGCCCCAAATACGAGGTTTGTTCGTTTGTTTTCAATATAATTATTTGCCACCAAGTGAGATATTACGGTTTTAAATAGCTTATTTGACGATACCAAACCCTGATAAGAGAAAGAAGAAAGAGAAAGCTGGTGTAGTTGTTTTAAGTGAACTAGATAAAAAATTAATAGCAAAACTTGAAAAAGATGGTGTGAAAATATCAAAAGAAGATGTTATAGGAATAAAATAATTGCCAGATGATGAGAAATCGTTTGGCTGGAAAAAGGAAATCCATCCGCTGGATTTGAGCATATTCTTATTGAACATGGTGAACAATTTGCTAAATAGGGAATTTCAAAAGCTGAGTTACCTGATTTTTTGATGACTGCTTTAGAAAAGGAAA >F-locusATTCTTTAAAAATATCTAATAATTTATTTACTATATACTCTAATACATCTTTTAACCTATCTAAAACATCATCACCTACAACATCCCAAAAATCATCTAAAAAGTTAAAAAAATCCATCTTTATCAACTCCTATATCTATTTTTTATTGTGTAATTCCTGAGTTACAAAACCATTATAACACGTATTACACACGTAGTCAATACTTCAAAAAAATTTTTTGTATATTTTTTTGAATAAGTAAATAAAAAGAGCTGTGTAGCTCTTTATTAAAATCAATATTTTTATTTTGTTAACAAACTTAGACAACATTAAATTTAGAAACCTATATATATTTCAGTACTTTTCATTTTTAGGTAGTCTAAATCAGAAATGGTTTTGTCTAAATGATGTATGTAAGTTTTAGTCCCCTTCGTTTTTAGGGTAGTCTAAATCAGAAGTCATTTAATAAGGCCACTGTTAAAAGTTTTAGTCCCCTTCGTTTTTAGGGTAGTCTAAATCCCATCCAAATTATGGGATAATATGTTACTTTTTATTTTAATATTTGATTATTTATTGTTTTTTTACTGATTTAGATTACCCCTTTAATTTATTTTACCATATTTTTCTCATAATGCAAACTAATATTCCAAAATTTTTGTTTCTTTTCTTATGATCTTTTCTCCGATAGTTATTTCTCCAGATAAGATTTTCATTTTTTTGAATTGATCTTCTGTTAGAATTAATGTTCTTACTGATGAATTTTCTGGAACTATCATTGACAACTGATTTTCATAGGAAATTATTTTTTCTTTTGTGCTAGAACTTACAATGTATACTGATTTTTGTACCTGATAATATCCTTTTCTTATAATTTCTTTTCTAAATTTTGCATATTCTTTTTTTTCTTTTCCTGTTTGCATTGGAAAATCATACATTAGAATCCCTACATAATTAGTACTCATAATCCTCTATCCTTAACTCAGGAATTTCTACTTCTGACATTTCTCCTGTAAAATAATTTCTAATATTATCTAAAAAATAATCAATCACTTGAGCCAATTCATATTTTTTATTTTTCCAATAAACTTTTTGTGTTAATACCAATAACAATTTTTGTCTTAATGATTTATTCAAACTTACTTCTTCCTGTTGATTAAAATATACGATATAATCTACCATTGGACGAAATATTTCAATAATATCATCTGCAAAATTATAATTATTAAATTGTGAACTGTGATGTATTCCCAAACTTGGATGAAATCCTTTAGCCACAATTTTTGAAGAGATTAAGCTTCTCAAAACCATATACCCATAATTTAATGCCGAATTTGTCCCGTCTTCACCAAATCTCTTAAATTTTTTCCCAAAAAGTTCACCAAAATACATTCTTGCAGCAATTGCTTCCTGATGTTCCGCTTCTTTTCCTTTTAATCTAATATTATTTTCATATGCTTCCAACTTATATGATACTTCCTGAGATTTTTTCAAAAACTGCAATAAATTTCTTTGATTTTCTATTTTTCTCATTACAATTTTTCTCCAGATTTCTTCTTTTTTATCGTCAATCCAGCTCACTTGCTCATTAATTCTTGTTGTTACTTGAAAATGATTATACAGTCCTAATGAATGTAAAACTGGCTGATGTTTTTCATTACAAATTATCAGTGGAATATTATGTTCTGATAATCTTAACTGTAATATTCCGCTAATTTTACATCTGCAATTTTCAACTACAATTGCCATGATATCATTTAAAGATACTTTATCAGCCTTATTTTCATCATCTTCATTTATCATCACAAGCTGGTTATTTAAAACTGATAATTCATTGACTCTTGTTACATGGATAATATTAGACATTTTTATTACTCCTTTACTCTAAAGCTTTATATTCAAACATAACTTTCACAAGTTCACACAATTCTTCTGAATTTCTATCAGTCATTAATTTTTTCTTTTTTAAATTTTTCAAATGTACAATTTTTTCCGATTCTAAAGTCTGAATTTCTATTTTCTTATCTGCTCCTATTTTAAATGTTGCTACAAAACCATATTCCTTTAATATATCCACTATTGATTTCATAATTGCATTTTTAAGTTTTCTATCATAAGAAAGTAATTTTCTTAAATTTTCCAGCACTTCTAAAAGTGAAATTTCAGCATGCGGAATATAGTTAAAATGTGCAATATAGTTTCGTATATACAAATCTTTTTTCTCTTGTTTTAATTTTTTTACTTTTTTATCAGAATAGATGCTTCTTTTTTCTACATTATCTTTGTATAATTCTTTATAAAAATTTATATATTTTTCAACAATTTGCCCACTTTTATATTTTACATTTTTACTGTTATCAAAATTAAATATTTCTTCAATATAATGATTTTCAGGAAATTCACCTTTCAATCTAAATCTTAAGTCCCTTTCCCAGATCGAAGTATATCCCACAAGTCTGTGGAGTATTTTTAATAACAAGCCTTGCAACAAGTTTAATTCATTAAATTCCACTTTATTTTTCAAATGAGTATATTTTTGTATATTTCCAATTGCTTTTTCATATTCTTTATAATCTTCATCATTAAATTTTTCATCTTTTTTAGGTCTTGCATATTTTCTATGTAAATTTTGCTGCATTGTATAATTTTTTTCTATTTCATTTTTTTTATTGCTGTATTCTTTCAATTCTTTTAAACTTATTTTATACTTCGCTTTATCAGCTATTTTTTCAAGTAAATTTAACATCCCATATTTTTTTATATTATAAAAAGCTCTATGCTTTATAATATTTTCTCCATCAAAATATATTTTATTTGTGTCAAATTTCTTCAATTCTTTCCTATCTTTTATTTTATTTTCATTAAAATCTAAAAATTTTCCAATTTCATTCGCTTCTAATTCAAAATCTTCTGTTACTCTATTATTATCTAAATTTAAAAGATTTATAAGTTCAAGTTCATCTGAAAAAGTTTCTTCTTTATTTGCACTCTGATATTTTTCAAGACTTCCCTTCAAATTAGTCAATTCTTTATGATTAAGCAATTTTAAAATTAAATAAAACATATTCAAATTTTCAGTGTATTTTAATATCTTTCCTAATTTTATCTCTCTTACAAATTCATTTATTTCATGTGGAATTTCTTTATTCCTATTATGTTTTTCATAATTTTTTAAAATTTTATCATATTTTTCTTTATTATCTTTTTTTATTTTTATTTTAGAAAATATATCATTATTATCATTGTTATTATTACTTTCTATATATTTTAAATTATTTTTATTCAAATAATCTATAAAACCTTTTAAAAATATTTGTTGTATAAAATCAATGTATGTATTTTTTTCTTCTTTATCTTGATTATTAATCATCTCCCTACTTTGTATAATAGCAAGATATTCTACTGGTACAGTTTTTTCTATATTTTCAAATTTTTGATATTTATAATGTCCTGTTTTTTGATTTCTTTGTTTATTTATTTTTATTACTTCATTAGTTATTTTAAAAAAAACTTTACTATTTTTAACAAATTTATTAAGAAATTCACCATAATAAATATTTTTCAAAAGATATATTTGAGCATCTTTTTCTTCTTTATCCTTAGGAACACTCCAAAAAAATTTTAAAGTATTTCTTAAATCTTCTATTTTATTATATAATTTCGTAAAAGAAGGAACAAAAGGAATATTCTTATTTACAAAATTAAATTTTGTATTTTTTAAATATTTAATTATCACATCCTTTTCATAATAATTAAATACATTTGCACTATTTAACTGCTTAAATATCTTCAATTTCAATTTTTTCTCATTTATTTCATTTTGAAACATTTTTTTTGAAATTTCAGAAGGAGCTATATTTTTAAATGCAAATATATCTTTCCCTTCTAATTCCAAATTAAAATGCACAATCCCATGTCTAATACTGCTAATAGCTTCATCAATATTTGCAAAAAAATCTTCTATCTCATTTTTATTATCCATATTAAAATCATAACTATAGAACATTTTTAAATTTTCTTTTACTTCATTTTGCTTGTTTTCATTATATATTTTATCAACTTCTCCAGAAACATATTTTTCTTCGCCCTTATTATTTTTTACAGTTTTTCCTCTCATTCTACCTGTAATATCATTCTCATTTTCAGTTTCAAGAATATTTCTCAATGAAAAATATGCAACCGAAGAAACTCCAATTATATTTCGTAAAAATGCTTCATTTTGTCTATTCCTAGCAATAAAATCACTTGTTGCAATCTCTCCAACTTGTAAATAATAATTGTATTTCCCACAATTTCTTACATAAGTATCCAATTTATTTAGTAATTTGTTTTCAATTAATTTTTTTAAATTTTGATATTCAAATATTCTCTTAATTTTATCGTTACTTATGTTACTCAGTCTTTTATACACATAATTTTTCAAAAGCTGACTCATTTCAATTTCCACAAAATGACAAAAAGCATATTTTATATTTTTATCATTAAGTTCTTCTTTATCCAAATAATATTTATAAAACACTTGTGATTTTTTTAATTCACTCATATCCGGAATTTTTTCAATTAATTCTTTTATATTATTTACATTTTGTATTTCTTCGTAAATAATTTTAGCAAAATTTTCTTTATCATTTTTTCTTCCAATTATTTTGTGATAGTATTCTCTTATTTTATATTTTTCATGTTTTTTTGAATTTTCTATTAAAAAAAATAACTTCTCAATATCTTCTTTTTTATACAATTTATCAAATGCTTCCTGTACATTATTTATATAATCATTACGCTTTGCTGATTCTCTATAATAATCATAAATAATATTTCTTTTGCTCTTCCCTCCAACTTTTTCAACATTATTTTCATTAATTTTCTGATAATTAGCCTTATTTTCTTCAAATGAATATTTTAAAGAATTTATCTTATTCAATTTTGCCTCAACATCTTTTCTAAATATTTCTAATTCTTCAGAGTTCACATCTTCATTTAACAATATTTTCTTTAAAACTGAAAAACTATTTTTATTTTTTAAATCATATTCTGAAATATCTTCTTCAGAATAATTTTTATCCTGTACTGCATTTTTCTCTTTCCTATTCTTTAAATACAGAACACTATCTTTTAGATGCAATACTTTATTTGAAAAAAACTTTTTTAAATTTTCTCTTCTTATTCTATTTTCTTCTTCACTTGCATTATCAGGATTTTTTATATATATATCCAGTCTTATACTTAAAAGCTCTGACAATCTCTCACTAGTCCTATTTTCTTCGCTCGTACTTTTTACTAATTTTCCCTCTTCAATATATTTTTTATGCGAAATTCCATCAACTTTTGTAACTTTCATATATAAAAACCTCCTAATATCTATATTTTTTACTCAATACCTAATTCTTTTTTCAATGCTTTTTGTAAAATTTGTGAAAAATTCAGATTTTTTTCCTGTGCCAATATATCTAACCAAACAGGAATTGTTAAAGTTTTCTTTTTAAGTGCATTTGTAACTTTTGCCACTTCATACACTGGATCAACAGATAAAATATACAAATACTGATTTTCTTTCAGTTTCACATCCTCCACTTTTGAAGGCTCAGGAAATTTTTTTCTTACATCCAAAAAATCAGCCAAATGCAGACCCAATGTCTCTCTCAAATTGGAAACAGCCTCCTCCATGCTATCTCCAAATGTAGCATAATAATTTATCTCTCCATCTTCAAACTTATCAAAATCAACAATACAACCATAATAAGTCCCATCTTCCTTAGTTACCACTGCTGGATAAAATACATCCATTTTAATTATCTCCAATCTATACCACGTGTTAAATACGTGTTTAAAAATATTTATAAAATTTTTTAGCATCTCTGCTAAAATAAAACAATTATTTCAAATTTTTCTATTCCTTAATCACTCATTGTTAGTGATTCTTTTTTTACTTGGACAATTTTTCATTTAATTTCTTCAATTTTTTTAAAATCACATTTTTTTAATATTCCTTATTTAATTGCAAATTTTCATTACTTTTGGGGTGCTCTAAATCCCATCCAAATTATGGGATAATAATTTTTAGTGAAAGCAAGAAGGGACTAGAATTTAATCCCAACTTGTTTTTCAATACTTCTTAATGTTCCTACAGGTATATCTTTTGAATATGGTACTGTGACCACACCTTCCACACCTGGGATCATCCATTGATAATGACTACCTCTTATACGCACAACTTTTCCGCCTAATTTTCTAAATCTTTTTTCGAT >G-locusCTTTCTATCTTTTTCAAATAAAATTAGGCTCTAGTTAGCCTAATCGCATAATTATTTATTATAGTATAATTCTTATTTTTTTTCAACCTAAAAATTTAAAACATCTCCAAAAATTTTCGTTTCAGAACAACCAAGCAACCATATTCAAAAAACAATAAAAAATGAGCAAGAATTGAAATTTTATTCTCACTCAGAAGTTATTTTTATTAAATATCACTTTTCGATATTGGGGTGGTCTATATCAATTTAAAAGACAGAATAGATAATTCTTTAGAGTTTTAGTCCCCTTCGATATTGGGGTGGTCTATATCAGAAGTCATTTAATAAGGCCACTGTTAAAAGTTTTAGTCCCCTTCGATATTGGGGTGGTCTATATCCCATCCTAATTTCTTGCTGATGAGATATTTATTTCTAATTTTTCTATTTTGTCTTTATTTTCAATACTTTCAATCCTATTTTTCTCTTTATTAATAATATAGAACCACCCTATACTATTATACCATATTTTTTGATTTTTCAAAATTCCAATATTTTGTTTTGTGAAATTTTTTCTCCCATTGTCACTTCTCCTGCAAGTACCTTCATTTTTTGAAACTGATCTTCTGTCAGGATAATGGAACGGATTGATGAATTTTCTGGAGCGAGCATTGATAACTGTTTTTCTGCCAGTTCGATTTTTTCTTTTGTTTTCGACCTCATTATATATACCGATTTTTGAAGCTGATAATATCCCTTTTCTATCAATTTTTTCCTAAAAGTCCTATATTCAAATCTCTCAACATCTGTCTGCATAGGAAAATCATACATAAGCAGACCAAAATACTCAATACTCATAGTCCATCACGCTCAATGTCGGAATTATCACTTCTTCATCTTTTACAAAATAATTTCGTATACTATCCAAATAATAGTCTACCGCTTGGAAAAAATCATATTTCTTATTGTTAAATAATACCTTCTGCTGTGCTACAAGAAGTATTTTTTGCCTTATTTCCTTACTTAATTTCACTTCATTCAAAATATCCTTGTACATATAAACAAGATAATCCACCATAGGACGAAAAACCTCTATTATATCATCAGAAAAATTATAGGCATTAAACTGTGACTTATGATGTAATCCTAAACTTGGATGAAATCCTTTTGCTACAATCTTTGATGATATTATAGCTCTTAAAATCATATATCCATAATTAAGTGCAGAATTCACTCCATCTTCATCAAATCTTTTAAAACTATTACTATACAATTCCTGAAAATATATCCTTGAAGCTATTGCTTCCTGATGTTCTGCACTCGCATCATCTTTTTTCAAGTTTTCCTTATATGTTTTCAGTCTTTCAATGGAAATATCACTTTTTTCAAGATACTCTAACAATGCTCTTTGATTTTCAATCTTATTCTCCACTATCCTGCTCCACAATTTTTCCTTTTTCTCTTTTTCCCACTCAATCTGCTCATTTATTCGTAAAGTCACTTGAAAATGATTAAATAATCCCAGCGAATGAATTTCAGGCTGATGTTTCTCGTTGCAAATAATAATCGGAATGTTATTTTCCACCAGCCTCAACTGCAAAATCGCACTAATCTTACAATAGCAGTTTTCAATAACTATCGCAGATATATCATTCAAAGAAATCTTATTTTTCTCATCATTATTGTCTTCATCAACCATTATAAGCTGATTATTCGATATTGACAAATCATCAGCCCTTGTTATGTGAATTATATTGGGCATTTTAATCATACTCCTTATAAATTTCATTCTTATAACGTATCATTCGTATTTTCTATTTTTGTTAAAAGTTCTATTATCAAGTTTTTAATATAATCAGAATTATAACTTTCTAATTCTAAAACAGAAACTTTTTTAGGTTTCATTAATCTTTCAAGTATATCATTATTACCGATAAGTTTAAATTTTTTCTTTAATTCATCATAATCTAAATTCACATCTTTTTTAAATACTTCAAATACACTTGCATAAGTTGAATTATTATAACGTGTACTATATGATAATAAATTAGAAACTCTATCAATTTGTTCTGCAATACTGTAATCAGCAAACGGATTTCTTACAATATAGAAATGTGAAATATAGTTTCTAATACTTTCATTTTCCGGCTTATTAATTTCAGAATTTTCAGACAAATCAATTCCAAATCCATAACATATTTTCTCAAATTTTTTATAAGATTCTTCATCAAAAAATTTATAGTATGCTGTTGTTGTATAAAAGCCATCAGATCCATTACGCTTAGGATAAGCTCTACTTATTCCAGTATTGTAGCCACTTAACTTAATAATTCCTAATTCTCTTAGCCCATTTACAATATAGTGCATATCTCTTTCAAATCTAGCCATTTGAATAGCAAGTTTCCAATTTATATCTATCAAATAACTTTCTATTTTATTCAAATAATTAAATTCTACCAAATCTCTAATTTTTTTGTATTCAGAAACTCTATTATAATCTTTTTCAAATGATTTATAGTTTTTATTTTGTATATTTTTTGCAAAAAAGTCATCATTTTCTTTCAATTTTTTTATATACTTCTCTTTGTATTCTTTAGAATATCCATTTAGTTTATCATTTAGATTTTTCAATATTGCATCAATTTCAGATATTTTATTTTTTCTAATATTTTTACCATCAATATTAAATAAAAATTTTGCATCAGCCATTTTAATATCATTTGAAATTAATCCATAAATTTTATCAAAATTTGGATTTCCAATATTTAAAAATAAATTCTTTTTATAAATATATAATTCATTCTTACGTTCTTTAGGATAATATATTTCTTGAAATTTATTTTCATTCTCTGATTCCATATCTTCTATTAAATTATCTATTTCTTTTTTGTATTTTTTTAAAAAATCAGAATTAAATATTATTCTACACAATATTTTACTCTTTATTTCCTGATCTTTATCTTTTATATACTGATCAACCTTTTTTTTCAAATCCTTTTTATTTATGTTTGATAACTTTCTTTGTTCATCTTGTAATATATTCGATTTTTTATCTATCTCAAATTTAGTTTCATCATCAAAAATTACAATTTTTTCTAATTTTTTCTCTAAAACATCACAACCATTAATATCATCTTTAAATTCAGTTAATATATTATTTTTTATATCCTCATAATAATTATTAAAAATTTCTTTTTTAGTTTGTATTTTAAAATCATCAAAGTCTTTTTCTATCTCTTTCATTTTTTGAATAAATTCTTCTAAATTAAGATTCCAATTTTCAGTTATACATTCATTTCTCAAAGTATTTAATTGCATTATTTCATCTAAAATATCTATAATATTTTGATATTCTGAAGTATTTAACCAAACTGATGTTGCAAAAAATCTATTTCTAATTTTATTTATAACCGCATTACTATTTAACAGTGCAAATATTGAAATTATATATTCAAAATCATCATTTATTACTATAGTTTTATCACTAGTCTTTACAGTTATTCTTTCGTAAGTTTTATTATCATTAATGTCTTTTATTTGTTTCTTAATTTCTTGAATATTCATTTTAAAATCTGAAAAATCAAAAAGTTCCTCATAATTTTTTCTCAAATATCCAATATAACATTCTATTACTTTTTTCTGATATTTTTTAATAGCTTTATTATTACCTTTTGAAGCAGAAATCTGAGCATTTTTATAATAATTTTCTATAATATTTTCATCTATTTCATCAATGTTTCCTAAAGTTTTCTTTAATTCTTGTAAAAATATATTCTTACTTTCATTTTCTTCTAAATCATCTTCTAAAATTAATTTCTTATACAATTCTTTATTCACATATATTAAAGCATTTAATACTATTTTTTCTGTTTCTATAGTATCAAATGGTTCATTCTTAGGATTATTCCTATATAAATTTAATATTTCAGGAAGTACTTTAGAAAAGGATGGTAAATATTTAATATCATTATTATTTTCTTCTGAAATTTTAATATCATTTATTTTAGTAATTATATTTTTTTTATCTTTAAATACTACATCTAAATTTAATGCTTTTGACACTTCTTCATCTGATATTTTTAAATTTTGAATTATATTTATGACTTTATTATAGTCATCTTGCGTTCCTTGTAAATCTCTTTCCTTGCTAATCGCATGTAATATCCTGTTTCTTTCATTTGTTCCTATCTTTGTAAATTTCCTAATAAAATTATTTGTAATGTTATTTTTATTATCTATAAAATCTAAGTCTCTTATTATTTTTATTTTTGAATTTAAAATTTTTTTATCAAGTACGTAATTTTTTTCTCGATCTCCTCCAAAGAAATCTATATTTTCATCATTATTTATATTTTCTCTAGAAAAAATCTTATTTAATTCCATATTGGTAGAAGCAAAAAAAGTAATCAATTCTAAATCCAATTCCTCTTTAGCGTGAAGTCTAGAAAAATCATCAGTATTTACTGTTGTCATATCTATATCATTATGTCTTAATTTCCCTAAATACATAATATGCTCTAACGTATATTGCTTAACTCTTTTTAAAATTTTTTCAGATAATATACTTTCATTTAAAATTTTTTCTATTTCTATTTTTTCCATTTTCTTTAATCTGACTTTTTGTTCATTTACCAATATTTTTTCAATTCTTCCTTTCAAATATCGATATATGATTTTATATAGTTCTTTTTCTTCATCAGATTTCTTTGAAAATTTTTTCGAATCAAAATTAACTTTATAATGTTTTTTAAATATTCCAAAAATTTCTGTATCACAATTTCCTTTTTTTAGTTCTTTTTCTAATTTTTTTATTAATTCATCTATTTTAAATTCTGCTAAAATTTTTTCTATTTTTTCTTTTATACTATTATTTTTTATATTTTCTACAAAAAATTTTACAATTTTATCTTTTTTATTTTCTCTTTCTATTTTAAATTTTTCGTGCTTATCTAATAGTACATAAGATTTTATATATGTTCTATTTCTTCTCTTTTCAAGAAATTCATTATTAACTTTTTTTACTTTTTCAATTCTTTTAGTAATATTCCAAAATTCTAACTCTTTTATAACAAAATCAGCTATATCTTCTACTGTTAAATCTACATTTATATTTAAAATTTTTTCAACAAGCATTTTTTTATTTTTAGATTTCTTTTTATCACCACCAACATTAAGATAAAATTTTACAAAACCCAGAATTTCTAAATTACTTTTTATTTTTTCTCTTATTTCCATAAAATTAGTCAAAATAACATCTATTTTATCATCTTTCAATAATTTTTCTCTTAAATGTTCTTCATAATATCGATTTTCAAATACTTTTTCTGTTTCATTTTCAATTATTTTTTCTATAATCTTATATAAACTCATGTTAATATTTTTAAAAATTTCGTAAATTGATTTTTTTGTTTCTAATTCATCATTTTCTATTATTCTTAATATTATTGAACAATCATTTAGTGTTTTATTAGTATACTCATCTCTGATATCTATCTCTATTTCTTCTTCATTCTCTTGTCTCTTTATTTCTATTTTTTTATCATCTTTAGTTATTCCTTGCCTAATTGCTTCATCTATTATTTTCTTTTTTGTAATCCCCAATGCTTTCAATTTCTCAGATTTTCCATATGCTTCTATATATAATACAACTTCTTCTGTTTCCAAAAAATCATCATTATTTTCTATTCTTATGATTCCTTCTTTACCTTTCAACTTAAATAGAATATTTCCTGCATGAAATTTTCTTGTAAATTCTTTAAGAATATTATCATTTTTTTTGTAATTAATATATTTTCTAATAAATTTATTATTATCAATTTTTTCTTTATTATTATTTTCATTAATATTTAAAATGTATTTGTTTCCATCATAGTTCCTTTTAACTTTTACTTTCCGTTTTATTTTAAAATCTTTTTTATCACGAACTTCATACCATCTCTTATGTCCAAATAAATTTCCCATTCCAATCTCCTCGTTTCTACTTTAATCTAATAAAATATTTTTAAATTAAATCAATTTTACATCTTTCTAATCAAAAATACAATTTTCCATTTTTAGTATACCACATCAATATTAAATCTCAAAAAAATAAGGAGCCGTCAAACATAGCTCCCTACTTCTATTTACTCATAATCCCCATCTATCCTTACTTTTCGTAAAATCAATCCTTCTTTCGCCTTTAGATCCAACTTAATTTTCCCATTTGAACCTGTTCTAAATGTTCTGCCTTCTGTTACCAAATCAATAAATCTTTCATCCTGATAATTTGTTTCAAATTCCACATTTTCCCAGCTGTTAAACGAATTATTTATTACAACAATAATTAAATGATCCTCGATTACTCTTTCATACACAATTATTT

Example 3: Further Evaluation of C2c2p and Associated Components

Applicants isolated both C2c2 loci from Carnobacterium gallinarum (FIG.46) and have studied domain structure and organization as well asexpression of the CRISPR array. At the first C2c2 locus, there is lowexpression of the two CRISPR arrays in the direction of the C2c2 gene.(See FIG. 47). The second locus also has low expression with directionof transcription in the direction of the C2c2 gene. (See FIG. 48).Applicants determined that both loci may have minimal expression sinceneither locus has associated Cas genes (Cas1/Cas2). Such associatedgenes may be needed for a functional CRISPR locus. Applicants haveoptimized methods to obtain C2c2 loci from other bacterial strains.

Applicants perform RNA-sequencing on the following strains that aregrown: Clostridium aminophilum DSM10710, Carnobacterium gallinarumDSM4847, Leptotrichia wade F0279, Leptotrichia shahii DSM19757, andRhodobacter capsulatus SB 1003.

Applicants design pACYC cloning from the following sources of DNA.Applicants clone the entire C2c2 locus into this backbone for DNA/RNAcutting experiments in E. coli.

1) Isolated genomic bacterial DNA: Lachnospiraceae bacterium MA2020,Lachnospiraceae bacterium NK4A179, Lachnospiraceae bacterium NK4A1442) From growing the strain: Clostridium aminophilum DSM10710,Carnobacterium gallinarum DSM4847, Leptotrichia wade F0279, Leptotrichiashahii DSM19757, Rhodobacter capsulatus SB1003

Applicants design PAM bait libraries for DNA cutting to evaluate thecutting ability of the C2c2p effector protein. Applicants design RNAcutting experiments based on the cutting of a resistance genetranscript.

Applicants test for function in mammalian cells using U6 PCR products:spacer (DR-spacer-DR) (in certain aspects spacers may be referred to ascrRNA or guide RNA or an analogous term as described in thisapplication) and tracr for following strains: Lachnospiraceae bacterium,Listeria seeligeri serovar 12b, Leptotrichia wadei, Leptotrichia shahii.Applicants mammalian codon optimized the DNA encoding the C2c2 proteinand cloned it into a plasmid from Genscript.

Applicants analyzed a representative C2c2 locus, i.e., the Listeriaseeligeri serovar ½b str. SLCC3954 C2c2 (LseC2c2) CRIPSR locus.Applicants performed RNA-sequencing on the L. seeligeri C2c2 locus whichwas cloned into E. coli. The LseC2c2 locus was synthesized by Genscriptinto a pET-28 vector. Cells harboring plasmids were made competent usingthe Z-competent kit (Zymo). E coli containing heterologous constructswere cultured in Luria broth supplemented with appropriate antibioticsin suspension at 37° C. and 300 rpm. The bacteria were grown in aerobicconditions and harvested in stationary growth phase.

RNA was isolated from stationary phase bacteria by first resuspendingthe bacteria in TRIzol and then homogenizing the bacteria withzirconia/silica beads (BioSpec Products) in a BeadBeater (BioSpecProducts) for 7 one-minute cycles. Total RNA was purified fromhomogenized samples with the Direct-Zol RNA miniprep protocol (Zymo),DNase treated with TURBO DNase (Life Technologies) and 3′dephosphorylated with T4 Polynucleotide Kinase (New England Biolabs).rRNA was removed with the bacterial Ribo-Zero rRNA removal kit(Illumina). RNA sequencing libraries were prepared from rRNA-depletedRNA using a derivative of the previously described CRISPR RNA sequencingmethod (Heidrich et al., 2015, Methods Mol Biol, vol. 1311, 1-21).Briefly, transcripts were poly-A tailed with E coli Poly(A) Polymerase(New England Biolabs), ligated with 5′ RNA adapters using T4 RNA Ligase1 (ssRNA Ligase), High Concentration (New England Biolabs), and reversetranscribed with AffinityScript Multiple Temperature ReverseTranscriptase (Agilent Technologies). cDNA was PCR amplified withbarcoded primers using Herculase II polymerase (Agilent Technologies).

The prepared cDNA libraries were sequenced on an MiSeq (Illumina). Readsfrom each sample were identified on the basis of their associatedbarcode and aligned to the appropriate RefSeq reference genome using BWA(Li and Durbin, 2009, Bioinformatics, vol. 25, 1754-1760). Paired-endalignments were used to extract entire transcript sequences using Picardtools (http://broadinstitute.github.io/picard) and these sequences wereanalyzed using Geneious 8.1.5.

The Applicants observed a high level of expression of the locus and theformation of small crRNAs with a 5′ 29-nt DR and 15-18-nt spacers (FIG.49A). Although the LseC2c2 locus contains a predicted putative tracrRNA(FIG. 15), the Applicants did not observe its expression (FIG. 49A).These findings suggest that the secondary structure present in thepre-crRNA of the LseC2c2 locus could be sufficient for processingyielding the mature crRNA as well as crRNA loading onto the C2c2protein. The RNA-folding of the processed crRNA shows a stronglypredicted stem loop within the direct repeat that potentially couldserve as a handle for the C2c2 protein (FIG. 49A).

The Applicants also expressed the Leptotrichia shahii str. SLCC3954 C2c2locus in E. coli and analyzed its expression using Northern blotting.The procedure was performed essentially as described in Pougach andSeverinov, 2012 (Methods Mol Biol, vol. 905, 73-86). E. coli BL21 AIcells were transformed with the plasmid pACYCduet-1 containing underinducible T7 promoter Leptotrichia shahii cas operon and plasmid pCDF-1bcontaining the minimal CRISPR cassette with a single spacer. Total RNAwas extracted from 5 mL of E. coli cells induced with 1 mM arabinose/0.2mM IPTG and grown until OD₆₀₀ 0.8-1.0. The cells were lysed by 5-minutetreatment using Max Bacterial Enhancement Reagent followed by RNApurification with the TRIzol reagent (Thermo Fisher Scientific). 15 μgof total RNA were separated on a denaturing 8 M urea—12% polyacrylamidegel and electrophoretically transferred to Hybond-XL membrane (GEHealthcare) using a Mini Trans-Blot Electrophoretic Transfer Cell(Bio-Rad). The membrane was dried and then UV cross-linked. ExpHybhybridization solution (Clontech) was used for hybridization accordingto manufacturer's instructions for 1 hour at 40° C. with ³²P-end labeledoligonucleotide probes. The Applicants found that the CRISPR array isexpressed and processed into 44-nt crRNAs (FIG. 49B). The expression andcrRNA formation was thus demonstrated herein in at least two distinctC2c2 loci using independent methods.

The Applicants sought to predict potential tracrRNAs for the rest of theidentified C2c2 loci by searching for anti-repeat sequences within eachlocus. In many CRISPR-Cas loci, the repeat located at thepromoter-distal end of the CRISPR array is degenerate and has a sequencethat is clearly different from the rest of repeats (Biswas et al., 2014,Bioinformatics, vol. 30, 1805-1813). Such degenerate repeats weredetected in several C2c2 and C2c1 systems, allowing the Applicants topredict the direction of the array transcription. By integrating thisinformation, putative tracrRNAs for 4 of the 17 C2c2 loci and 4 of the13 C2c1 loci were identified. In some subtype II-B and II-C loci, theCRISPR array is transcribed in the opposite direction, starting from thedegenerate repeat (Sampson et al., 2013, Nature, vol. 497, 254-257;Zhang et al., 2013, Mol Cell, vol. 50, 488-503). Accordingly, weattempted to predict the tracrRNA in different positions with respect tothe CRISPR array but were unable to identify additional candidatetracrRNA sequences. Conceivably, the prediction of tracrRNA for otherloci was hampered by a combination of factors such as imperfectcomplementarity to repeats, lack of an associated CRISPR array, and/orpotential incompleteness of the loci. Furthermore, the possibilityremains that not all Class 2 CRISPR systems require tracrRNA.

The Applicants identified depleted sequence motifs in order to identifyPAM nucleotides. A PAM library was prepared in a bacterial vector andtransformed into a strain of E coli expressing LshC2c2 (FIG. 54). Infurther detail, the assay is as follows for a RNA target, provided thata PAM sequence is required to direct recognition. Two E. coli strainsare used in this assay. One carries a plasmid that encodes theendogenous effector protein locus from the bacterial strain. The otherstrain carries an empty plasmid (e.g. pACYC184, control strain). Allpossible 7 or 8 bp PAM sequences are presented on an antibioticresistance plasmid (pUC19 with ampicillin resistance gene). The PAM islocated next to the sequence of proto-spacer 1 (the RNA target to thefirst spacer in the endogenous effector protein locus). Two PAMlibraries were cloned. One has a 8 random bp 5′ of the proto-spacer(e.g. total of 65536 different PAM sequences=complexity). The otherlibrary has 7 random bp 3′ of the proto-spacer (e.g. total complexity is16384 different PAMs). Both libraries were cloned to have in average 500plasmids per possible PAM. Test strain and control strain weretransformed with 5′PAM and 3′PAM library in separate transformations andtransformed cells were plated separately on ampicillin plates.Recognition and subsequent cutting/interference with the plasmid rendersa cell vulnerable to ampicillin and prevents growth. Approximately 12 hafter transformation, all colonies formed by the test and controlstrains where harvested and plasmid RNA was isolated. Plasmid RNA wasused as template for PCR amplification and subsequent deep sequencing.Representation of all PAMs in the untransfomed libraries showed theexpected representation of PAMs in transformed cells. Representation ofall PAMs found in control strains showed the actual representation.Representation of all PAMs in test strain showed which PAMs are notrecognized by the enzyme and comparison to the control strain allowsextracting the sequence of the depleted PAM. CRISPR interference resultsin ineffective transformation by plasmids containing an effective targetsequence. Transformant plasmids were sequenced to identify non-targetsequences. Depleted sequences identify the 5′ PAM nucleotides (FIG. 55).Heterologous targeting in E. coli was observed for three targets.Increased interference was observed for more highly transcribed targets.In particular, a target in a transcribed region (“RNA)” coincided withincreased interference compared to minimally transcribed target “DNA1”and “DNA2” (FIG. 56). A 5′ DNA PAM screen showed no DNA cleavage (FIG.85A-B). LshC2c2 does not cleave untranscribed or transcribed DNA in anE. coli RNAP in-vitro assay (FIG. 83). LshC2c2 does not cleave ssDNA ordsDNA in vitro (FIG. 84).

LshC2c2 components were purified for in vitro tests (FIG. 57).Initially, it was observed in test reactions that crRNA is cleaved byC2c2. Cleavage of crRNA is not Mg²⁺ dependent, and may be elevated inthe absence of target (FIG. 58). It was further found that there isreduced cleavage in absence of Mg (FIG. 108)

After showing the capability of the LshC2c2 CRISPR locus to mediatessRNA interference, we wanted to demonstrate two additional aspects ofC2c2 activity: 1) RNA interference using an orthogonal assay, and 2) theability to retarget C2c2 to endogenously expressed transcripts in acell. We developed a fluorescent readout for LshC2c2 activity byexpressing RFP from a transfected plasmid in E coli (FIG. 63A). We thendesigned three spacers for each of the three possible H PAMs (9 spacerstotal) targeting the RFP mRNA and cloned them into the pLshC2c2 backboneas before. We transfected these plasmids into E. coli already expressingthe RFP plasmid and grew them under double selection over night. Byanalyzing the RFP levels in E. coli by flow cytometry, we observedrobust RFP knockdown for all three PAMs and no RFP knockdown for spacerstargeting the anti-sense DNA strand or non-targeting spacers (FIG.63B-C). To further investigate LshC2c2 targeting and cleavge activity,spacers targeting RFP were cloned into the LshC2c2 locus and the locuswas expressed in E. coli carrying a plasmid encoding expressible RFP ora pUC19 control plasmid. FIG. 61 shows C2c2 targeted the transcribedRFP. Strand-dependency of interference was investigated by selectingtarget sequences coinciding with or complementary to transcribedregions. High levels of interference were observed using targetingsequences complementary to transcribed RNA (FIG. 62). The extent ofinterference was also observed to vary among transcribed targets,possibly in relation to transcription levels (FIG. 63A-63C).

RNA targeting and target selection was investigated using a modeltranscript and testing different targets (FIG. 65). Cleavge of RNA wastested using RNA targets (FIGS. 66 and 67) and RNA transcribed from DNAtemplates (FIGS. 68 and 69). Observed RNA cleavage products coincided insize with those expected from the model transcript (FIG. 70). FIG. 87demonstrates that LshC2c2 does not require the small RNA for RNAcleavage.

Example 4: C2C2 Targets and Cuts RNA In Vitro

Leptrichia shahii C2c2 is Capable of Interference Against ssRNA MS2Phage

C2c2 was first discovered in a computational search of conserved unknownproteins near the adaptation protein Cas2 in order to uncover novelClass 2 CRISPR systems (Shmakov et al) and is hypothesized to be thefunctional effector of a novel CRISPR sub-type VI group because oflittle homology to other known CRISPR proteins. The C2c2 proteins hastwo conserved HEPN domains that show strong conservation of the activeresidues but little homology to any other known HEPN superfamilyproteins or CRISPR effectors. However, C2c2 differs from other HEPNproteins, particularly CRISPR-associated type III proteins Csx1 andCsm6, which typically dimerize prior to cleavage of RNA, because it hastwo HEPN domains rather than one. Many of these unique features haveprompted the classification of C2c2 as a putative type VI. Given theseobservations and the prevalence of C2c2-family proteins across diversebacterial species, we sought to determine whether C2c2 CRISPR-Cas lociare biologically active and can mediate interference against RNA.

To determine whether the Leptotrichia shahii C2c2 (LshC2c2) is afunctional RNA-targeting system, we cloned the entire LshC2c2 CRISPR-Caslocus into low-copy plasmids (pLshC2c2) to allow heterologousreconstitution in E. coli. With currently characterized DNA- andRNA-targeting CRISPR systems, target cleavage is dependent on twofactors: 1) complementarity between the crRNA spacer sequence and targetsite (protospacer) and 2) the presence of the appropriate proximaladjacent motif (PAM) flanking the protospacer. Because the PAMrequirement is meant to discriminate self vs. non-self recognition, itis unclear whether a uniquely RNA-targeting system would require a PAMsince presumably there would be no self-RNA to even target.

To investigate the PAM requirements and activity of LshC2c2, we used theMS2 phage restriction assay (FIG. 73). MS2 phage is an ideal model toinvestigate RNA cleavage because it is a lytic single-stranded RNA phagethat has no DNA intermediates during its life cycle. It readily infectsE coli via attachment to the F pilus and can thus be used for testingheterologous interference against ssRNA. We synthesized a library ofcrRNA sequences to tile every possible 28nt target site in the MS2 phagegenome in order to identify which target sites were more significantlydepleted than others. The crRNA library was cloned into pLshC2c2 suchthat each unique spacer was the first spacer of a two-spacer array. Wetransformed this library into E. coli NovaBlue (DE3, F+) and grew thecultures with or without MS2 phage overnight. Using this assay, we wereable to identify a single-nucleotide PAM by analyzing the flankingregions of the crRNA target sequences that were enriched due toresistance against MS2 infection. The analysis revealed a 3′ H PAM (notG) on the RNA target indicative that there is some sequence preferenceby the LshC2c2 complex (FIG. 75 A-B). Beyond the identification of aPAM, the screen revealed that the heterologously expressed LshC2c2 locuswas capable of significant ssRNA interference and protection against MS2phage infection.

To validate the screen findings, we cloned four of the top enrichedspacers and showed 3- to 4-log reduction in plaquing efficiencyconsistent with the level of enrichment observed in the screen.Moreover, we wanted to further validate the PAM finding and so we cloneda series of four guides per possible single nucleotide PAM (16 guides intotal) all targeting a region of the MS2 mat gene. We found that all 16targets were efficiently targeted with a stronger preference for C, A,and U. Because G PAMs are still targeted and there were a minorityenriched in the interference screen, the PAM may be more relaxed than a3′ H PAM.

The C2C2 protein from leptotrichia shahii was expressed in E. coli andpurified using His-tag affinity purification followed by three rounds ofgel filtration on an Akta FPLC using a Superdex 200 column. For in vitrocleavage experiments, a 175 nucleotide RNA target (labeled t1 and t3respectively, see below for sequences) was combined with a 5× molarexcess of C2c2 protein and crRNA (using a 28 nucleotide spacer and a 28nucleotide direct repeat, see below for sequence) and incubated at 37Cfor 15 minutes in the buffers indicated in the figure panels. Thereaction was quenched with proteinase K incubation for 15 min at 37C andsubsequently denatured in TBE-Urea loading buffer at 85C for 5 min.Samples were resolved on a denaturing TBE Urea PAGE gel.

The results indicate that C2C2 mediates efficient degradation of the RNAtarget in a crRNA-dependent manner. (FIG. 71, FIG. 79, FIG. 80) Notably,the crRNA itself is also cleaved during this process.

Targeting of RFT transcripts in bacteria showed that growth rate isreduced (FIG. 77). Without wishing to be bound by theory, this maysuggest that the HEPN system is a suicidal phage defense system.

Cleavage fragments were mapped as indicated in FIGS. 81 and 114A-B-2.

RNA-sequencing of IVC (in vitro cleavage) was performed as indicated inFIG. 82.

RNA target 1 sequence aatatggattacttggtagaacagcaatctaCGCCAGTGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTttatccgctcacaattccacacaacatacgagccggaagcataaag RNA target 3 sequenceGGCCAGTGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAaatatggattacttggtagaacagcaatctaCTCGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTttatccgctcacaattccacacaacatacgagccggaagcataaag crRNA sequenceCCACCCCAATATCGAAGGGGACTAAAACtagattgctgttctaccaagta atccatL. shahii C2c2protein sequenceMGNLFGHKRWYEVRDKKDFKIKRKVKVKRNYDGNKYILNINENNNKEKIDNNKFIRKYINYKKNDNILKEFTRKFHAGNILFKLKGKEGIIRIENNDDFLETEEVVLYIEAYGKSEKLKALGITKKKIIDEAIRQGITKDDKKIEIKRQENEEEIEIDIRDEYTNKTLNDCSITLRIIENDELETKKSIYEIFKNINMSLYKIIEKIIENETEKVFENRYYEEHLREKLLKDDKIDVILTNFMEIREKIKSNLEILGFVKFYLNVGGDKKKSKNKKMLVEKILNINVDLTVEDIADFVIKELEFWNITKRIEKVKKVNNEFLEKRRNRTYIKSYVLLDKHEKFKIERENKKDKIVKFFVENIKNNSIKEKIEKILAEFKIDELIKKLEKELKKGNCDTEIFGIFKKHYKVNFDSKKFSKKSDEEKELYKIIYRYLKGRIEKILVNEQKVRLKKMEKIEIEKILNESILSEKILKRVKQYTLEHIMYLGKLRHNDIDMTTVNTDDFSRLHAKEELDLELITFFASTNMELNKIFSRENINNDENIDFFGGDREKNYVLDKKILNSKIKIIRDLDFIDNKNNITNNFIRKFTKIGTNERNRILHAISKERDLQGTQDDYNKVINIIQNLKISDEEVSKALNLDVVFKDKKNIITKINDIKISEENNNDIKYLPSFSKVLPEILNLYRNNPKNEPFDTIETEKIVLNALIYVNKELYKKLILEDDLEENESKNIFLQELKKTLGNIDEIDENIIENYYKNAQISASKGNNKAIKKYQKKVIECYIGYLRKNYEELFDFSDFKMNIQEIKKQIKDINDNKTYERITVKTSDKTIVINDDFEYIISIFALLNSNAVINKIRNRFFATSVWLNTSEYQNIIDILDEIMQLNTLRNECITENWNLNLEEFIQKMKEIEKDFDDFKIQTKKEIENNYYEDIKNNILTEFKDDINGCDVLEKKLEKIVIFDDETKFEIDKKSNILQDEQRKLSNINKKDLKKKVDQYIKDKDQEIKSKILCRIIFNSDFLKKYKKEIDNLIEDMESENENKFQEIYYPKERKNELYIYKKNLFLNIGNPNFDKIYGLISNDIKMADAKFLFNIDGKNIRKNKISEIDAILKNLNDKLNGYSKEYKEKYIKKLKENDDFFAKNIQNKNYKSFEKDYNRVSEYKKIRDLVEFNYLNKIESYLIDINWKLAIQMARFERDMHYIVNGLRELGIIKLSGYNTGISRAYPKRNGSDGFYTTTAYYKFFDEESYKKFEKICYGFGIDLSENSEINKPENESIRNYISHFYIVRNPFADYSIAEQIDRVSNLLSYSTRYNNSTYASVFEVFKKDVNLDYDELKKKFKLIGNNDILERLMKPKKVSVLELESYNSDYIKNLIIELLTKIENTNDTLKRPAATKKAGQAKKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYA

RNA PAM screen using MS2 phage interference identified PAMs, see FIGS.73-78D. It was determined that LshC2c2 has a 3′ PAM for RNA cleavage(FIG. 86). FIGS. 88 and 89 also demonstrate that LshC2c2 isreprogrammable and PAM sensitive.

FIG. 90A-90B demonstrates that LshC2c2 cannot use spacers less than18-22nt.

FIGS. 91A-93D show the influence of stem loop (modifications) oncleavage. It is shown that crRNAs without stem loop do not allow forcleavage (FIG. 91A-C) and that Stem is amenable to individual base swapsbut activity is disrupted by secondary structure changes. DR Truncationexperiments also indicate that disruption of the stem abolishes cleavage(FIG. 92A-D and FIG. 93A-D).

FIG. 100A-100B shows the effects of divalent cations on C2c2 activity.

It was shows that C2c2 cuts 3′ of the target site (FIG. 102 and FIG.103)

FIGS. 104-106 show that C2c2 can be reprogrammed with crRNAs.

Without wishing to be bound by theory, it seems that one active, C2c2becomes active and degrades other RNAs (FIG. 98; FIG. 108).

It was shown that C2c2 may be reprogrammed with crRNAs (FIGS. 104-106),and that long targets may be targeted (FIG. 107)

FIG. 112A-112D suggests that C2c2 crRNAs have a seed region, asindicated by single and double mismatch analysis. Indeed, doublemismatch in nt 1-11 of target significantly affects cleavage while lessso if in region region spanning nt 16-26. These figures also demonstratespecificity of cleavage of C2c2 effector protein.

FIG. 113-1-113-3 and FIG. 114A-114B-2. Suggest that changing sequencecontexts affects cleavage patterns. Indeed target sequences provided indifferent context are cleaved differently.

Example 5: Mutation of Either HEPN Domain Abolishes Tageted Cleavage

Cpc2 variants were created comprising the mutations R597A and R1278A. Asshown in FIG. 72, both mutations abolished RNA cleavage, see also FIG.97A-97D, demonstrating that R597A, H602A, R1278A, and H1283A abolish RNAcleavage

HEPN mutants however still process natural array (FIG. 95).

HEPN mutants still retain targeted binding activity, as demonstrated byFIG. 111 (EMSA analysis). Top panel: binding of wild type C2c2. Bottompanel: binding of R1278A mutated Lsh C2c2.

Example 6

Corresponding residues in other C2c2 orthologs were identified bystructural alignment to identify structural representatives thatcorrespond to either their experimentally determined structures orhomology models. FIG. 109-1-109-2 illustrates the sequences alignment ofthe following orthologs of the Leptotrichia shahii DSM 19757 C2c2;Rhodobacter capsulatus SB 1003 (RcS); Rhodobacter capsulatus R121 (RcR);Rhodobacter capsulatus DE442 (RcD); Lachnospiraceae bacterium MA2020(Lb(X)); Lachnospiraceae bacterium NK4A179 (Lb(X); [Clostridium]aminophilum DSM 10710 (CaC); Lachnospiraceae bacterium NK4A144 (Lb(X);Leptotrichia wadei F0279 (Lew); Leptotrichia wadei F0279 (Lew);Carnobacterium gallinarum DSM 4847 (Cg); Carnobacterium gallinarum DSM4847 (Cg); Paludibacter propionicigenes WB4 (Pp); Listeria seeligeriserovar ½b (Ls); Listeria weihenstephanensis FSL R9-0317 (Liw); andListeria bacterium FSL M6-0635 (Lib). FIG. 110 demonstrates that C2c2orthologues have conserved HEPN domains.

Using the numbering from a consensus sequence obtained using MUSCLEalignment (www.ebi.ac.uk/Tools/msa/muscle/), the following conservedresidues were identified. K36, K39, V40, E479, L514, V518, N524, G534,K535, E580, L597, V602, D630, F676, L709, I713, R717 (HEPN), N718, H722(HEPN), E773, P823, V828, I879, Y880, F884, Y997, L1001, F1009, L1013,Y1093, L1099, L1111, Y1114, L1203, D1222, Y1244, L1250, L1253, K1261,I1334, L1355, L1359, R1362, Y1366, E1371, R1372, D1373, R1509 (HEPN),H1514 (HEPN), Y1543, D1544, K1546, K1548, V1551, I1558. The pairwisematch up of conserved residues in the consensus sequence with aminoacids of Leptotrichia wadei C2c2 (sequence F herein) is: K36, K2; K39,K5; V40, V6; E479, E301; L514, L331; V518, I335; N524, N341; G534, G351;K535, K352; E580, E375; L597, L392; V602, L396; D630, D403; F676, F446;L709, I466; I713, I470; R717 (HEPN), R474; N718, H475; H722 (HEPN),H479; E773, E508; P823, P556; V828, L561; I879, I595; Y880, Y596; F884,F600; Y997, Y669; L1001, I673; F1009, F681; L1013, L685; Y1093, Y761;L1099, L676; L1111, L779; Y1114, Y782; L1203, L836; D1222, D847; Y1244,Y863; L1250, L869; L1253, I872; K1261, K879; I1334, I933; L1355, L954;L1359, I958; R1362, R961; Y1366, Y965; E1371, E970; R1372, R971; D1373,D972; R1509 (HEPN), R1046; H1514 (HEPN), H1051; Y1543, Y1075; D1544,D1076; K1546, K1078; K1548, K1080; V1551, I1083; I1558, I1090.

Example 7: Generation of C2c2 Mutants with Enhanced Specificity

Recently a Method was Described for the Generation of Cas9 Orthologswith Enhanced specificity (Slaymaker et al. 2015). This strategy can beused to enhance the specificity of C2c2 orthologs. Primay residues formutagenesis are all positive charges residues within the HEPN domain,since this is the only known structure in the absence of a crystal andwe know that specificity mutants in RuvC worked in Cas9. The conservedArginine residues within HEPN domain are R717 and R1509.

Additional candidates are positive charged residues that are conservedbetween different orthologs. such as K2, K39, K535, K1261, R1362, R1372,K1546 and K1548. These can be used to generate C2c2 mutants withenhanced specificity.

Example 8: C2c2 is a Single-Component Programmable RNA-GuidedRNA-Targeting CRISPR Effector

TABLE A crRNA sequences used for in vitro experiments. Name Sequence1st FIG. crRNA14 CCACCCCAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAA 117BGUAAUCCAU crRNA15 CCACCCCAAUAUCGAAGGGGACUAAAACUUUCUAGAGGAUCCCCGGG 117DUACCGAGCU crRNA16 CCACCCCAAUAUCGAAGGGGACUAAAACAGUAAUCCAUAUUUCUAGA 117DGGAUCCCCG crRNA17 CCACCCCAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAA 117DGUAAUCCAU crRNA18 CCACCCCAAUAUCGAAGGGGACUAAAACCAUGCCUGCAGGUCGAGUA 117DGAUUGCUGU crRNA19 CCACCCCAAUAUCGAAGGGGACUAAAACGCAUGCCUGCAGGUCGAGU 117DAGAUUGCUG crRNA20 CCACCCCAAUAUCGAAGGGGACUAAAACAAGCUUGCAUGCCUGCAGG 117DUCGAGUAGA crRNA21 CCACCCCAAUAUCGAAGGGGACUAAAACCGCCAAGCUUGCAUGCCUG 117DCAGGUCGAG crRNA22 CCACCCCAAUAUCGAAGGGGACUAAAACGAUUACGCCAAGCUUGCAU 117DGCCUGCAGG crRNA23 CCACCCCAAUAUCGAAGGGGACUAAAACUGAUUACGCCAAGCUUGCA 117DUGCCUGCAG crRNA24 CCACCCCAAUAUCGAAGGGGACUAAAACAUGACCAUGAUUACGCCAA 117DGCUUGCAUG crRNA25 CCACCCCAAUAUCGAAGGGGACUAAAACUAUGACCAUGAUUACGCCA 117DAGCUUGCAU crRNA26 CCACCCCAAUAUCGAAGGGGACUAAAACAGCUAUGACCAUGAUUACG 117DCCAAGCUUG crRNA27 CCACCCCAAUAUCGAAGGGGACUAAAACGAAACAGCUAUGACCAUGA 117DUUACGCCAA crRNA28 CCACCCCAAUAUCGAAGGGGACUAAAACACAGGAAACAGCUAUGACC 117DAUGAUUACG crRNA29 CCACCCCAAUAUCGAAGGGGACUAAAACAACACAGGAAACAGCUAUG 117DACCAUGAUU crRNA30 CCACCCCAAUAUCGAAGGGGACUAAAACAAACACAGGAAACAGCUAU 117DGACCAUGAU crRNA31 CCACCCCAAUAUCGAAGGGGACUAAAACAUAAACACAGGAAACAGCU 117DAUGACCAUG crRNA32 CCACCCCAAUAUCGAAGGGGACUAAAACGGAUAAACACAGGAAACAG 117DCUAUGACCA crRNA33 CCACCCCAAUAUCGAAGGGGACUAAAACAGCGGAUAAACACAGGAAA 117DCAGCUAUGA crRNA34 CCACCCCAAUAUCGAAGGGGACUAAAACGAGCGGAUAAACACAGGAA 117DACAGCUAUG Lsh_crRNA_DR_28CCACCCCAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAA 120B GUAAUCCAULsh_crRNA_DR_26 ACCCCAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAAGU 120BAAUCCAU Lsh_crRNA_DR_24 CCCAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAAGUAA120B UCCAU Lsh_crRNA_DR_22CAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAAGUAAUC 120B CAU Lsh_crRNA_DR_20AUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAAGUAAUCCA 120B U Lsh_crRNA_DR_19UAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAAGUAAUCCAU 120B Lsh_crRNA_DR_18AUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAAGUAAUCCAU 120B Lsh_crRNA_24CCACCCCAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAA 120A GUAAU Lsh_crRNA_23CCACCCCAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAA 120A GUAA Lsh_crRNA_22CCACCCCAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAA 120A GUA Lsh_crRNA_21CCACCCCAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAA 120A GU Lsh_crRNA_20CCACCCCAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAA 120A G Lsh_crRNA_19CCACCCCAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAA 120A Lsh_crRNA_18CCACCCCAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCA 120A Lsh_crRNA_17CCACCCCAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACC 120A Lsh_crRNA_16CCACCCCAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUAC 120A Lsh_crRNA_12CCACCCCAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUU 120A Lsh_stem_1CCACCCGAAUAUCGAACGGGACUAAAACUAGAUUGCUGUUCUACCAA 121A GUAAUCCAULsh_stem_2 CCACCGCAAUAUCGAAGCGGACUAAAACUAGAUUGCUGUUCUACCAA 121AGUAAUCCAU Lsh_stem_3 CCACGCCAAUAUCGAAGGCGACUAAAACUAGAUUGCUGUUCUACCAA121A GUAAUCCAU Lsh_stem_4CCAGCCCAAUAUCGAAGGGCACUAAAACUAGAUUGCUGUUCUACCAA 121A GUAAUCCAULsh_stem_5 CCAGGGGGAAUAUCGAACCCCACUAAAACUAGAUUGCUGUUCUACCA 121AAGUAAUCCAU Lsh_stem_6 CCACCACCAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCA121A AGUAAUCCAU Lsh_stem_7CCAACCCAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAA 121A GUAAUCCAULsh_stem_8 CCACCCAAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAA 121AGUAAUCCAU Lsh_stem_9 CCACCCCCAAUAUCGAAGGGGGACUAAAACUAGAUUGCUGUUCUACC121A AAGUAAUCCAU Lsh_loop_1CCACCCCAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAAG 121B UAAUCCAU Lsh_loop_2CCACCCCAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAAGUA 121B AUCCAU Lsh_loop_3CCACCCCAAGGGGACUAAAACUAGAUUGCUGUUCUACCAAGUAAUCC 121B AU Lsh_loop_4CCACCCCAAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCA 121B AGUAAUCCAULsh_loop_5 CCACCCCAAAAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUAC 121BCAAGUAAUCCAU Lsh_loop_6 CCACCCCAAAAAAAAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUU121B CUACCAAGUAAUCCAU Lsh_loop_7CCACCCCGAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAA 121B GUAAUCCAULsh_loop_8 CCACCCCAAAAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAA 121BGUAAUCCAU Lsh_loop_9 CCACCCCAAUAUCCAAGGGGACUAAAACUAGAUUGCUGUUCUACCAA121B GUAAUCCAU Lsh_single_mismatch_pos1CCACCCCAAUAUCGAAGGGGACUAAAACAAGAUUGCUGUUCUACCAA 122C GUAAUCCAULsh_single_mismatch_pos5 CCACCCCAAUAUCGAAGGGGACUAAAACUAGAAUGCUGUUCUACCAA122C GUAAUCCAU Lsh_single_mismatch_pos9CCACCCCAAUAUCGAAGGGGACUAAAACUAGAUUGCAGUUCUACCAA 122C GUAAUCCAULsh_single_mismatch_pos13CCACCCCAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUGUACCAA 122C GUAAUCCAULsh_single_mismatch_pos17CCACCCCAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACGAA 122C GUAAUCCAULsh_single_mismatch_pos21CCACCCCAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAA 122C GAAAUCCAULsh_single_mismatch_pos25CCACCCCAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAA 122C GUAAUGCAULsh_single_mismatch_pos28CCACCCCAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAA 122C GUAAUCCAALsh_double_mismatch_pos1 CCACCCCAAUAUCGAAGGGGACUAAAACAUGAUUGCUGUUCUACCAA122D GUAAUCCAU Lsh_double_mismatch_pos6CCACCCCAAUAUCGAAGGGGACUAAAACUAGAUACCUGUUCUACCAA 122D GUAAUCCAULsh_double_mismatch_pos11CCACCCCAAUAUCGAAGGGGACUAAAACUAGAUUGCUGAACUACCAA 122D GUAAUCCAULsh_double_mismatch_pos16CCACCCCAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUAGGAA 122D GUAAUCCAULsh_double_mismatch_pos21CCACCCCAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAA 122D GAUAUCCAULsh_double_mismatch_pos26CCACCCCAAUAUCGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAA 122D GUAAUCGUU

TABLE B ssRNA targets used in this study. Name Target 1st FIG.ssRNA 1 (C PAM) GGCCAGUGAAUUCGAGCUCGGUACCCGGGGAUCCUCUAGAAAUAUGGAUU 117BACUUGGUAGAACAGCAAUCUACUCGACCUGCAGGCAUGCAAGCUUGGCGUAAUCAUGGUCAUAGCUGUUUCCUGUGUUUAUCCGCUCACAAUUCCACACAACAUACGAGCCGGAAGCAUAAAG ssRNA 2AAUAUGGAUUACUUGGUAGAACAGCAAUCUACAAAAAAAAAAAAAAAAAA 118BAGAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAAAAAA AG ssRNA 3AAUAUGGAUUACUUGGUAGAACAGCAAUCUACUUUUUUUUUUUUUUUUUU 118BUCUUUUUUUUUUUUUUUUUUUCUUUUUUUUUUUUUUUUUUUCUUUUUUUUUUUUUUUUUUUCUUUUUUUUUUUUUUUUUUUCUUUUUUUUUUUUUUUUUU UC ssRNA 4GGGUAGGUGUUCCACAGGGUAGCCAGCAGCAUCCUGCGAUGCAAAUAUGG 118CAUUACUUGGUAGAACAGCAAUCUAAUCCGGAACAUAAUGGUGCAGGGCGCUGACUUCCGCGUUUCCAGACUUUACGAAACACGGAAACCGAAGACCAUUCAUGUUGUUGCUGCCGGAAGCAUAAAG ssRNA 5GGGCCCCUCCGUUCGCGUUUACGCGGACGGUGAGACUGAAGAUAAUAUGG 118CAUUACUUGGUAGAACAGCAAUCUAAACUCAUUCUCUUUAAAAUAUCGUUCGAACUGGACUCCCGGUCGUUUUAACUCGACUGGGGCCAAAACGAAACAGUGGCACUACCCCGCCGGAAGCAUAAAG ssRNA 1 (G PAM)GGCCAGUGAAUUCGAGCUCGGUACCCGGGGAUCCUCUAGAAAUAUGGAUU 117CACUUGGUAGAACAGCAAUCUAGUCGACCUGCAGGCAUGCAAGCUUGGCGUAAUCAUGGUCAUAGCUGUUUCCUGUGUUUAUCCGCUCACAAUUCCACACAACAUACGAGCCGGAAGCAUAAAG ssRNA 1 (A PAM)GGCCAGUGAAUUCGAGCUCGGUACCCGGGGAUCCUCUAGAAAUAUGGAUU 117CACUUGGUAGAACAGCAAUCUAAUCGACCUGCAGGCAUGCAAGCUUGGCGUAAUCAUGGUCAUAGCUGUUUCCUGUGUUUAUCCGCUCACAAUUCCACACAACAUACGAGCCGGAAGCAUAAAG ssRNA 1 (U PAM)GGCCAGUGAAUUCGAGCUCGGUACCCGGGGAUCCUCUAGAAAUAUGGAUU 117CACUUGGUAGAACAGCAAUCUAUUCGACCUGCAGGCAUGCAAGCUUGGCGUAAUCAUGGUCAUAGCUGUUUCCUGUGUUUAUCCGCUCACAAUUCCACACAACAUACGAGCCGGAAGCAUAAAG ssRNA 6ACCGAUCGUCGUUGUUUGGGCAAUGCACGUUCUCCAACGGUGCUCCUAUG 124BGGGCACAAGUUGCAGGAUGCAGCGCCUUACAAGAAGUUCGCUGAACAAGCAACCGUUACCCCCCGCGCUCUGAGAGCGGCUCUAUUGGUCCGAGACCAAUGUGCGCCGUGGAUCAGACACGCGGU ssRNA 7ACUGUUGGUGGUGUAGAGCUUCCUGUAGCCGCAUGGCGUUCGUACUUAAA 124BUAUGGAACUAACCAUUCCAAUUUUCGCUACGAAUUCCGACUGCGAGCUUAUUGUUAAGGCAAUGCAAGGUCUCCUAAAAGAUGGAAACCCGAUUCCCUCAGCAAUCGCAGCAAACUCCGGCAUCU ssRNA 8GGUAACAUGCUCGAGGGCCUUACGGCCCCCGUGGGAUGCUCCUACAUGUC 124BAGGAACAGUUACUGACGUAAUAACGGGUGAGUCCAUCAUAAGCGUUGACGCUCCCUACGGGUGGACUGUGGAGAGACAGGGCACUGCUAAGGCCCAAAUCUCAGCCAUGCAUCGAGGGGUACAAU ssRNA 9UUCGUAAAACGUUCGUGUCCGGGCUCUUUCGCGAGAGCUGCGGCGCGCAC 124BUUUUACCGUGGUGUCGAUGUCAAACCGUUUUACAUCAAGAAACCUGUUGACAAUCUCUUCGCCCUGAUGCUGAUAUUAAAUCGGCUACGGGGUUGGGGAGUUGUCGGAGGUAUGUCAGAUCCACG ssRNA 10AUAGGCCAGUGAAUUCGAGCUCGAAUAUGGAUUACUUGGUAGAACAGCAA 119DUCUACGCCGGAAGCAUAAAG ssRNA 10(rc)CUUUAUGCUUCCGGCGUAGAUUGCUGUUCUACCAAGUAAUCCAUAUUCGA 119DGCUCGAAUUCACUGGCCUAU ssDNA targetctttatgcttccggctcgtatgttgtgtggaattgtgagcggataaacac 119Daggaaacagctatgaccatgattacgccaagcttgcatgcctgcaggtcgagaatatggattacttggtagaacagcaatctatctagaggatccccgggtaccgagctcgaattcactggccccctatagtgagtcgtattaatttc

TABLE C Spacers used for in vivo experiments. Name Sequence 1st FIG.spacer 1 GAAGUUUGCAGCUGGAUACGACAGACGG 1116D spacer 2UGUCUGGAAGUUUGCAGCUGGAUACGAC 1116D spacer 3 AGCUGGAUACGACAGACGGCCAUCUAAC1116D spacer 4 UACGUCGCGAUAUGUUGCACGUUGUCUG 1116D spacer 5UACGGACGACCUUCACCUUCACCUUCGAUUU 123A spacer 6UCGUACGGACGACCUUCACCUUCACCUUCGA 123A spacer 7CGGUCUGGGUACCUUCGUACGGACGACCUUC 123A spacer 8GCGGUCUGGGUACCUUCGUACGGACGACCUU 123A spacer 9AGCGGUCUGGGUACCUUCGUACGGACGACCU 123A spacer 10AGUUCAUAACACGUUCCCAUUUGAAACCUUC 123A spacer 11UUAACUUUGUAGAUGAACUCACCGUCUUGCA 123A spacer 12UUUAACUUUGUAGAUGAACUCACCGUCUUGC 123A spacer 13GUUUAACUUUGUAGAUGAACUCACCGUCUUG 123A spacer 35AAGUUUGCAGCUGGAUACGACAGACGGC 119B spacer 36 ACAGGAUGUCCCAAGCGAACGGCAGCGG139 spacer 37 GCUUGUUCAGCGAACUUCUUGUAAGGCG 129A spacer 38UAAGCUCGCAGUCGGAAUUCGUAGCGAA 129A spacer 39 CGUCAACGCUUAUGAUGGACUCACCCGU129A spacer 40 UCAACAGGUUUCUUGAUGUAAAACGGUU 129A spacer 41AAGUUUGCAGCUGGAUACGACAGACGGC 130 spacer 42 UUUGCAGCUGGAUACGACAGACGGCCAU130 spacer 43 CAGCUGGAUACGACAGACGGCCAUCUAA 130 spacer 44GUUGUCUGGAAGUUUGCAGCUGGAUACG 130 spacer 45 GCGAUAUGUUGCACGUUGUCUGGAAGUU130 spacer 46 ACGUUGUCUGGAAGUUUGCAGCUGGAUA 130 spacer 47AUGUUGCACGUUGUCUGGAAGUUUGCAG 130 spacer 48 UGCAGCUGGAUACGACAGACGGCCAUCU130 spacer 49 CUGGAAGUUUGCAGCUGGAUACGACAGA 130 spacer 50AGUUUGCAGCUGGAUACGACAGACGGCC 130 spacer 51 GCUGGAUACGACAGACGGCCAUCUAACU130 spacer 52 GUUUGCAGCUGGAUACGACAGACGGCCA 130spacer_41_single_mismatch_pos1 UAGUUUGCAGCUGGAUACGACAGACGGC 122Aspacer_41_single_mismatch_pos5 AAGUAUGCAGCUGGAUACGACAGACGGC 122Aspacer_41_single_mismatch_pos9 AAGUUUGCUGCUGGAUACGACAGACGGC 122Aspacer_41_single_mismatch_pos13 AAGUUUGCAGCUCGAUACGACAGACGGC 122Aspacer_41_single_mismatch_pos17 AAGUUUGCAGCUGGAUUCGACAGACGGC 122Aspacer_41_single_mismatch_pos21 AAGUUUGCAGCUGGAUACGAGAGACGGC 122Aspacer_41_single_mismatch_pos25 AAGUUUGCAGCUGGAUACGACAGAGGGC 122Aspacer_41_double_mismatch_pos1 UUGUUUGCAGCUGGAUACGACAGACGGC 122Bspacer_41_double_mismatch_pos6 AAGUUACCAGCUGGAUACGACAGACGGC 122Bspacer_41_double_mismatch_pos11 AAGUUUGCAGGAGGAUACGACAGACGGC 122Bspacer_41_double_mismatch_pos16 AAGUUUGCAGCUGGAAUCGACAGACGGC 122Bspacer_41_double_mismatch_pos21 AAGUUUGCAGCUGGAUACGAGUGACGGC 122BHeterologous Reconstitution of the L. shahii C2c2 Locus in Escherichiacoli Confers RNA-Guided Immunity Against a RNA Bacteriophage

As a first step, we explored whether LshC2c2 could be used to conferimmunity to MS2 (G. Tamulaitis et al., Programmable RNA shredding by thetype III-A CRISPR-Cas system of Streptococcus thermophilus. Mol Cell 56,506-517 (2014)), a lytic single-stranded (ss) RNA phage without DNAintermediates in its life cycle that readily infects E. coli. Weconstructed a low-copy plasmid carrying the entire LshC2c2 locus(pLshC2c2) to allow for heterologous reconstitution in E. coli (FIG.126). Given that expressed mature crRNAs from the LshC2c2 locus have amaximum spacer length of 28nt (FIG. 126) (S. Shmakov et al., Discoveryand Functional Characterization of Diverse Class 2 CRISPR-Cas Systems.Mol Cell 60, 385-397 (2015)), we synthesized a library of 3,473 spacersequences tiling all possible 28-nt target sites in the MS2 phage genomeand cloned them as spacers into the pLshC2c2 CRISPR array. Aftertransformation in E. coli, cells were infected with MS2 and spacersequences in cells that survived the infection were determined. Cellscarrying spacers that confer robust interference against MS2 willproliferate more rapidly, leading to enrichment of these spacersfollowing growth for 16 hours. A number of spacers were consistentlyenriched across two independent replicas, suggesting that they enabledstrong interference against MS2 (107 spacers showed >1.3 log 2-foldenrichment in both replicas; FIG. 116B and FIG. 127A-B). By analyzingthe flanking regions of protospacer on the MS2 genome corresponding tothe 107 enriched spacers, we found that spacers with G immediatelyadjacent to the 3′ end of the protospacer performed more poorly thanthose with an H (i.e. A, U, or C), indicating a single nucleotide PAM, H(FIG. 116C and FIG. 127C-D, FIG. 128A-128G).

To validate the interference activity of enriched spacers, weindividually cloned four top-enriched spacers into pLshC2c2 CRISPRarrays and observed a 3- to 4-log reduction in plaque formation,consistent with the level of enrichment observed in the screen (FIG.116B and FIG. 129A-129B). To confirm the PAM, we cloned sixteen guidestargeting distinct regions of the MS2 mat gene (4 guides per possiblesingle-nucleotide PAM). We found that all 16 crRNAs mediated MS2interference, although higher levels of resistance were observed for theC, A, and U PAM-targeting guides (FIGS. 116D, 116E and FIG.130-1-130-2), indicating that C2c2 can be effectively retargeted in acrRNA-dependent fashion to sites within the MS2 genome.

C2c2 is a Single-Effector endoRNase that Mediates ssRNA Cleavage with aSingle crRNA Guide

To test whether LshC2c2 mediated phage interference by facilitatingcrRNA-guided ssRNA cleavage, we purified the LshC2c2 protein (FIG.131A-131D) and assayed its ability to cleave an in vitro transcribed173-nt ssRNA target (FIG. 117A and FIG. 132A-132B) containing a C PAMprotospacer (ssRNA target 1 with protospacer 14). Previously, we foundthat mature LshC2c2 crRNAs contain a 28-nt direct repeat (DR) and a 28nt spacer (FIG. 126) (S. Shmakov et al., Discovery and FunctionalCharacterization of Diverse Class 2 CRISPR-Cas Systems. Mol Cell 60,385-397 (2015)), and we therefore generated an in-vitro-transcribedcrRNA with 28-nt spacer complementary to protospacer 14 on ssRNAtarget 1. We found that LshC2c2 efficiently cleaved ssRNA in a Mg2+- andcrRNA-dependent manner (FIG. 117B and FIG. 133). To investigate cleavageof dsRNA substrates, we annealed complementary RNA oligos to regionsflanking the crRNA target site. This partially double-stranded RNAsubstrate was not cleaved by LshC2c2, indicating it is specific forssRNA (FIGS. 134A-B).

To further characterize the sequence constraints of RNA cleavage byLshC2c2, we tested additional crRNAs complementary to different versionsof ssRNA target 1 where protospacer 14 is preceded by each PAM variant.The results of this experiment confirmed the preference for C, A, and UPAMs, with little cleavage activity detected for the G PAM target (FIG.117C). Additionally, we designed 5 crRNAs for each possible PAM (20total) across ssRNA target 1 and evaluated cleavage activity for LshC2c2paired with each of these crRNAs. As expected, we found less cleavageactivity for G PAM-targeting crRNAs compared to other crRNAs tested(FIG. 117D).

LshC2c2 was tested for DNA cleavage activity in vitro. We generated adsDNA plasmid library with protospacer 14 preceded by 7 randomnucleotides to account for any PAM requirements. When incubated withLshC2c2 protein and a crRNA complementary to protospacer 14, no cleavageof the dsDNA plasmid library was observed (FIG. 134C). We also did notobserve cleavage when targeting a ssDNA version of ssRNA target 1 (FIG.134D). To rule out co-transcriptional DNA cleavage which has beenobserved in type III CRISPR-Cas systems (P. Samai et al.,Co-transcriptional DNA and RNA Cleavage during Type III CRISPR-CasImmunity. Cell 161, 1164-1174 (2015)), we recapitulated the E. coli RNApolymerase co-transcriptional cleavage assay (P. Samai et al.,Co-transcriptional DNA and RNA Cleavage during Type III CRISPR-CasImmunity. Cell 161, 1164-1174 (2015)) (FIG. 135A), expressing ssRNAtarget 1 from a DNA substrate. Using this assay with the purifiedLshC2c2 and crRNA targeting ssRNA target 1, we still did not observe anyDNA cleavage (FIG. 135B). Together, these results indicate that C2c2cleaves specific ssRNA sites directed by the target complementarityencoded in the crRNA, with a 3′ H PAM requirement.

C2c2 Cleavage Depends on Local Target Sequence and Secondary Structure

Given that C2c2 did not efficiently cleave dsRNA substrates and thatssRNA forms complex secondary structures, we reasoned that cleavage byC2c2 might be affected by secondary structure of the ssRNA target. Intiling ssRNA target 1 with different crRNAs (FIG. 117D), the samecleavage pattern was observed regardless of the crRNA position along thetarget RNA, suggesting that the crRNA-dependent cleavage pattern wasdetermined by some features of the target sequence rather than thedistance from the binding site. We hypothesized that the LshC2c2-crRNAcomplex binds the target and cleaves exposed regions of ssRNA within thesecondary structure elements, with a potential preference for certainnucleotides. We analyzed the cleavage efficiencies of homopolymer RNAtargets (a 28-nt protospacer extended with 120 As or Us regularlyinterspaced by single bases of G or C to enable oligo synthesis) andfound that LshC2c2 preferentially cleaved the uracil target compared toadenine (FIG. 118A-B). To assess the impact of the target RNA on thecleavage pattern, we tested cleavage of three ssRNA targets withdifferent sequences flanking a constant 28-nt protospacer and foundthree distinct patterns of cleavage (FIG. 118C). RNA-sequencing of thecleavage products for the three targets revealed that cleavage sitesmainly localized to uracil-rich regions of ssRNA or ssRNA-dsRNAjunctions within the in silico predicted co-folds of the target sequencewith the crRNA (FIG. 118D-I).

The HEPN Domains of C2c2 Mediate RNA-Guided ssRNA-Cleavage

Previous bioinformatics analysis of C2c2 suggested that the HEPN domainsare potentially responsible for the catalytic activity we observed (S.Shmakov et al., Discovery and Functional Characterization of DiverseClass 2 CRISPR-Cas Systems. Mol Cell 60, 385-397 (2015)). Each of thetwo HEPN domains of C2c2 contains a dyad of conserved arginine andhistidine residues (FIG. 119A), in agreement with the catalyticmechanism of the HEPN endoRNAse (V. Anantharaman, K. S. Makarova, A. M.Burroughs, E. V. Koonin, L. Aravind, Comprehensive analysis of the HEPNsuperfamily: identification of novel roles in intra-genomic conflicts,defense, pathogenesis and RNA processing. Biol Direct 8, 15 (2013); O.Niewoehner, M. Jinek, Structural basis for the endoribonuclease activityof the type III-A CRISPR-associated protein Csm6. RNA 22, 318-329(2016); N. F. Sheppard, C. V. Glover, 3rd, R M. Terns, M. P. Terns, TheCRISPR-associated Csx1 protein of Pyrococcus furiosus is anadenosine-specific endoribonuclease. RNA 22, 216-224 (2016)). To testwhether these predicted catalytic residues were required for ssRNAdepletion in vivo, we mutated each residue separately to alanine (R597A,H602A, R1278A, H1283A) in the LshC2c2 locus plasmids and assayed for MS2interference. None of the four mutant plasmids were able to protect E.coli from phage infection (FIG. 119B and FIG. 136).

In order to validate these findings in vitro, the four single-pointmutant proteins were purified and assayed their ability to cleave5′-end-labeled ssRNA target 1 (FIG. 119C). In agreement with our in vivoresults, all four mutations abolished cleavage activity. The inabilityof either of the two wild-type HEPN domains to compensate forinactivation of the other implies cooperation between the two domains,which agrees with observations that several bacterial and eukaryoticsingle-HEPN proteins function as dimers (O. Niewoehner, M. Jinek,Structural basis for the endoribonuclease activity of the type III-ACRISPR-associated protein Csm6. RNA 22, 318-329 (2016); N. F. Sheppard,C. V. Glover, 3rd, R. M. Terns, M. P. Terns, The CRISPR-associated Csx1protein of Pyrococcus furiosus is an adenosine-specificendoribonuclease. RNA 22, 216-224 (2016); G. Kozlov et al., StructuralBasis of Defects in the Sacsin HEPN Domain Responsible for AutosomalRecessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS). J Biol Chem286, 20407-20412 (2011)).

Catalytically inactive variants of Cas9 retain target DNA binding,allowing for the creation of programmable DNA-binding proteins (G.Gasiunas, R. Barrangou, P. Horvath, V. Siksnys, Cas9-crRNAribonucleoprotein complex mediates specific DNA cleavage for adaptiveimmunity in bacteria. Proc Natl Acad Sci USA 109, E2579-2586 (2012); M.Jinek et al., A programmable dual-RNA-guided DNA endonuclease inadaptive bacterial immunity. Science 337, 816-821 (2012)). To determineif target binding and cleavage activity of LshC2c2 are likewiseseparable, electrophoretic mobility shift assays (EMSA) were performedon both the wild-type (FIG. 119D) and R1278A mutant LshC2c2 (FIG. 119E)in complex with crRNA. The wild-type LshC2c2 complex bound strongly (KD˜46 nM, FIG. 137A) and specifically to ssRNA target 10, but not to thenon-target ssRNA (the reverse complement of ssRNA target 10). The R1278Amutant C2c2 complex showed an even stronger (KD ˜7 nM, FIG. 137B)specific binding, indicating that this HEPN mutation results in acatalytically inactive, RNA-programmable RNA-binding protein. TheLshC2c2 protein or crRNA alone showed substantially reduced levels oftarget affinity as expected (FIG. 137C-E).

These results demonstrate that C2c2 cleaves RNA using a catalyticmechanism distinct from other known CRISPR-associated RNases. Inparticular, the type III Csm and Cmr multiprotein complexes rely onacidic residues of RRM domains for catalysis, whereas C2c2 achieves RNAcleavage through conserved basic residues of its two HEPN domains.

Sequence and Structural Requirements of C2c2 crRNA

Similar to the type V-B (Cpf1) systems (B. Zetsche et al., Cpf1 is asingle RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163,759-771 (2015)), the LshC2c2 crRNA contains a single stem loop in thedirect repeat (DR), suggesting that the secondary structure of the crRNAcould facilitate interaction with LshC2c2. To explore this possibility,we first investigated the length requirements of the spacer sequence forssRNA cleavage and found that LshC2c2 requires spacers of at least 22 ntlength to efficiently cleave ssRNA target 1 (FIG. 120A). We also foundthat the stem-loop structure of the crRNA is critical for ssRNA cleavagebecause DR truncations that disturbed the stem loop abrogated targetcleavage (FIG. 120B). Thus, a DR longer than 24 nt is required tomaintain the stem loop necessary for LshC2c2 to mediate ssRNA cleavage.

Next, we studied the effects of modifications in the stem and loop ofthe crRNA DR on the cleavage activity. Single basepair inversions in thestem that preserved the stem structure did not affect the activity ofthe LshC2c2 complex but inverting all four G-C pairs in the stemeliminated the cleavage despite maintaining the duplex structure (FIG.121A). Other perturbations that introduced kinks and reduced orincreased base-pairing in the stem also eliminated or significantlysuppressed cleavage, suggesting that the crRNA stem length is importantfor complex formation and activity (FIG. 121A). Through a series ofmodifications, we found that loop deletions eliminated cleavage, whereasinsertions and substitutions mostly maintained some level of cleavageactivity (FIG. 121B). Together, these results demonstrate that LshC2c2recognizes structural characteristics of its cognate crRNA but isamenable to loop insertions and most tested base substitutions. Theseresults have implications for the future application of C2c2-based toolsthat require guide engineering for recruitment of effectors ormodulation of activity (S. Kiani et al., Cas9 gRNA engineering forgenome editing, activation and repression. Nat Methods 12, 1051-1054(2015); S. Konermann et al., Genome-scale transcriptional activation byan engineered CRISPR-Cas9 complex. Nature 517, 583-588 (2015); J. E.Dahlman et al., Orthogonal gene knockout and activation with acatalytically active Cas9 nuclease. Nat Biotechnol 33, 1159-1161(2015)).

C2c2 Cleavage is Sensitive to Double Mismatches in the crRNA-TargetDuplex

We tested the sensitivity of the LshC2c2 system to single mismatchesbetween the crRNA guide and target RNA by mutating single bases acrossthe spacer to the respective complementary bases (e.g., A to U) andquantified plaque formation with these mismatched spacers in the MS2infection assay (FIG. 122A and FIG. 138). We found that C2c2 was fullytolerant to single mismatches across the spacer as such mismatchedspacers interfered with phage propagation with similar efficiency asfully matched spacers. However, when we introduced consecutive doublesubstitutions in the spacer, we found ˜3 log-fold reduction in theprotection for mismatches in the center, but not at the 5′- or 3′-end,of the crRNA (FIG. 122B and FIG. 138). This observation indicates thepresence of a mismatch-sensitive “seed region” in the center of thecrRNA-target duplex.

We further evaluated the requirements of LshC2c2 for the guide andtarget to match in vitro. To this end, we generated a set of in vitrotranscribed crRNAs with mismatches similarly positioned across thespacer region. When incubated with LshC2c2 protein, all singlemismatched crRNA supported cleavage (FIG. 122C), in agreement with ourin vivo findings. When tested with a set of consecutive double mutantcrRNAs, LshC2c2 was unable to cleave the target RNA if the mismatcheswere positioned in the center, but not at the 5′- or 3′-end of the crRNA(FIG. 122D), supporting the existence of a core seed region.

Sensitivity of the LshC2c2 system to double and triple mismatches wasalso evaluated. Double mismatches were spaced apart (FIG. 143A) whereastriple mismatches were consecutive (FIG. 143B). Cleavage sensitivity wasposition dependent. Mismatches proximal to the DR region did not supportcleavage whereas distal mismatches supported detectable cleavage.

The LshC2c2 system is also sensitive to mismatches and deletions in thedirect repeat region. Single mismatches and single base deletions weregenerally sufficient to disrupt ssRNA cleavage. Only one mismatch(mutant 7) supported a low level of cleavage activity (FIG. 144).

C2c2 can be Reprogrammed to Mediate Specific mRNA Knockdown In Vivo

Given the ability of C2c2 to cleave target ssRNA in a crRNAsequence-specific manner, we tested whether LshC2c2 can be reprogrammedto degrade selected non-phage ssRNA targets, and particularly mRNAs, invivo. To this end, we co-transformed E coli with a plasmid encodingLshC2c2 and a crRNA targeting the mRNA of red fluorescent protein (RFP)as well as a compatible plasmid expressing RFP (FIG. 123A). We observedan approximately 20% to 92% decrease in RFP positive cells for crRNAstargeting protospacers flanked by C, A, or U PAMs for OD-matched samples(FIG. 123B, 123C). As a control, we tested crRNAs containing reversecomplements (targeting the dsDNA plasmid) of the top performing RFPmRNA-targeting spacers. As expected, we observed no decrease in RFPfluorescence by these crRNAs (FIG. 123B). We also confirmed thatmutation of the catalytic arginine residues in either HEPN domain toalanine precluded RFP knockdown (FIG. 139). Thus, C2c2 is capable ofgeneral retargeting to arbitrary ssRNA substrates, governed exclusivelyby predictable nucleic-acid interactions.

When we examined the growth rate of cells carrying the RFP-targetingspacer with the greatest level of RFP knockdown, we noted that the ratewas significantly reduced (FIG. 123A, spacer 7). To determine the causefor this growth restriction, we investigated whether the effect ongrowth was mediated by the RFP mRNA-targeting activity of LshC2c2 byintroducing an inducible-RFP plasmid and an RFP-targeting LshC2c2 locusinto E. coli. Using this system, we found that upon induction of RFPtranscription, cells with RFP knockdown showed substantial growthsuppression, which was not observed in non-targeting controls (FIG.123D, 123E). However, in the absence of RFP transcription, we did notobserve any growth restriction nor did we observe anytranscription-dependent DNA targeting in our biochemical experiment(FIG. 135A-135B), which suggests that RNA-targeting is likely theprimary driver of this growth restriction phenotype. Without wishing tobe bound by theory, one possible explanation for this effect is thatC2c2 CRISPR systems might function to prevent virus reproduction byindiscriminately cleaving cellular mRNAs and causing reduced celldivision, programmed cell death (PCD) or dormancy (K. S. Makarova, Y. I.Wolf, E. V. Koonin, Comprehensive comparative-genomic analysis of type 2toxin-antitoxin systems and related mobile stress response systems inprokaryotes. Biol Direct 4, 19 (2009); F. Hayes, L. Van Melderen,Toxins-antitoxins: diversity, evolution and function. Crit Rev BiochemMol Biol 46, 386-408 (2011)).

C2c2 Cleaves Collateral RNA in Addition to crRNA-Targeted ssRNA

In contrast to Cas9 and Cpf1, which cleave DNA within the crRNA-targetheteroduplex at a defined position, reverting into an inactive stateafter cleavage, C2c2 cleaves the target RNA outside of the crRNA bindingsite at varying distances depending on flanking sequence, presumablywithin exposed ssRNA loop regions (FIG. 118D-I). This observedflexibility in cleavage distance lead us to consider the possibility ofcleavage of nearby non-target ssRNAs upon C2C2 target binding andactivation. Accordingly, C2c2 could cause PCD through a two-partmechanism: a priming stage in which C2c2-crRNA complexes bind to targetsites and cleave ssRNA in a crRNA-guided fashion and a second stage inwhich primed C2c2 cleaves non-targeted, collateral RNA non-specifically.To test this hypothesis, we carried out in vitro cleavage reactions thatincluded, in addition to LshC2c2, crRNA and its target RNA, one of fourunrelated RNA molecules without any complementarity to the crRNA guide(FIG. 124A). These experiments showed that, whereas the LshC2c2-crRNAcomplex did not mediate cleavage of any of the four collateral RNAs inthe absence of the target RNA, all four were efficiently degraded in thepresence of the target RNA (FIG. 124B and FIG. 140A). Furthermore, R597Aand R1278A HEPN mutants were unable to cleave collateral RNA (FIG.140B). These results indicate a HEPN-dependent mechanism whereby C2c2 ina complex with crRNA is activated upon binding to target RNA andsubsequently cleaves any nearby ssRNA targets. Such promiscuous RNAcleavage may cause cellular toxicity, resulting in the observed growthrate inhibition. These findings imply that, in addition to their role indirect suppression of RNA viruses, type VI CRISPR-Cas systems couldfunction as mediators of a distinct variety of PCD/dormancy inductionthat is specifically triggered by the cognate invader genomes (FIG.125). Under this scenario, dormancy would slow the infection and supplyadditional time for adaptive immunity to succeed; when adaptive immunityfails, the suicidal role of C2c2 would prevail and spread of theinfection would be limited. Such a mechanism falls within the previouslyproposed scheme of coupling between adaptive immunity and PCD during theCRISPR-Cas defensive response (K. S. Makarova, V. Anantharaman, L.Aravind, E. V. Koonin, Live virus-free or die: coupling of antivirusimmunity and programmed suicide or dormancy in prokaryotes. Biol Direct7, 40 (2012)).

Example 8: Expression of C2c2 in Eukaryotic Cells

A number of C2c2 orthologues were codon optimized for expression inmammalian cells using a mammalian expression vector. The various C2c2orthologues were transfected in HEK293T cells and cellular localizationwas evaluated based on mCerry expression. Cytoplasmic localization aswell as nuclear localization of the C2c2 protein was observed.

Example 9: Activity of C2c2 in Eukaryotic Cells

A luciferase targeting assay was performed with different gRNAs directedagainst the C2c2 protein. Efficient knockdown was observed.

A targeting assay based on GFP expression was performed with gRNAsdirected against EGFP. Expression of GFP was determined and compared tonon-targeting (NT) gRNA. Here too efficient knockdown was observed.

A targeting assay was performed on different endogenous target genes inHEK293 cells with gRNAs directed against endogenous target genes. C2c2.Expression protein expression of the respective target genes wasdetermined (compared to non-targeting (NT) gRNA). Efficient knockdown ofthe different target genes was observed.

Methodology for the Examples Cloning of C2c2 Locus and Screening Library

Genomic DNA from Leptotrichia shahii DSM 19757 (ATCC) was extractedusing the Blood & Cell Culture DNA Mini Kit (Qiagen) and the C2c2 CRISPRlocus was PCR amplified and cloned into a pACYC184 backbone withchloramphenicol resistance. For retargeting of the locus to MS2 phage orendogenous targets, the wild type spacers in the array were removed andreplaced with a Eco31I landing site an additional spacer and adegenerate repeat, compatible with Golden Gate cloning.

A custom library consisting of all possible spacers targeting the genomeof the bacteriophage MS2, excluding spacers containing the Eco31Irestriction site, was synthesized by Twist Biosciences, cloned into theretargeting backbone with Golden Gate cloning, transformed into EnduraDuo electrocompetent cells (Lucigen) and subsequently purified using aNucleoBond Xtra MaxiPrep EF (Machery-Nagel).

Bacterial Interference Assay

For the phage screen, 50 ng of the plasmid library were transformed intoNovaBlue(DE3) Competent Cells (EMD Millipore) followed by an outgrowthat 37° C. for 30 minutes. Cells were then grown in Luria broth (LB)supplemented with 25 μg/mL chloramphenicol (Sigma) in a volume of 4.5mL. Phage conditions were treated with 7*10 PFU of Bacteriophage MS2(ATCC). After 3 hours of shaking incubation at 37° C., samples wereplated on LB-agar plates supplemented with chloramphenicol and harvestedafter 16 hours. DNA was extracted using NucleoBond Xtra MaxiPrep EF(Machery-Nagel), PCR amplified, and sequenced using a MiSeq (Illumina)with a paired-end 150 cycle kit.

To determine enriched spacers, spacer regions were extracted, counted,and normalized to total reads for each sample. For a given PAM,enrichment was measured as the log ratio compared to input library, witha 0.01 psuedocount adjustment. PAMs above a 1.3 enrichment thresholdthat occurred in both biological replicates were used to generatesequence logos (G. E. Crooks, G. Hon, J. M. Chandonia, S. E. Brenner,WebLogo: a sequence logo generator. Genome research 14, 1188-1190(2004)).

To test individual spacers for MS2 interference, the oligos were orderedfrom IDT, annealed and phosphorylated with polynucleotide kinase (NewEngland Biosciences) and cloned into the locus backbone with Golden Gatecloning. Plasmids were transformed into C3000 strain E. coli, madecompetent with the Mix and Go kit (Zymo Research). C3000 cells wereseeded from an overnight culture grown to OD600 of 2, at which pointthey were diluted 1:13 in Top Agar and poured on LB-chloramphenicolplates. Dilutions of MS2 phage were then spotted on the plates using amultichannel pipette, and the creation of plaques was recorded afterovernight incubation.

RFP Targeting Assay

An ampicillin resistant RFP-expressing plasmid (pRFP) was transformedinto DH5-alpha cells (New England Biolabs). Cells containing pRFP werethen made chemically competent (Zymo Research Mix and Go) to be used fordownstream targeting experiments with pLshC2c2. Spacers targeting RFPmRNA were cloned into pLshC2c2 and these plasmids were transformed intothe chemically competent DH5-alpha pRFP cells. Cells were then grownovernight under double selection in LB and subjected to analysis by flowcytometry when they reached an OD600 of 4.0. Knockdown efficiency wasquantified as the percent of RFP positive cells compared to anon-targeting spacer control (the endogenous LshC2c2 locus in pACYC184).

To interrogate the effect of LshC2c2 activity on the growth of the hostcells, we created a TetR-inducible version of the RFP plasmid in pBR322(pBR322_RFP). We transformed E coli cells with this vector and then madethem chemically competent (Zymo Research Mix and Go) to prepare them fordownstream experiments. We cloned pLshC2c2 plasmids with various spacerstargeting RFP mRNA as well as their reverse complement controls andtransformed them into E. coli cells carrying pBR322_RFP and streakedthem on double-selection plates to maintain both plasmids. Colonies werethen picked and grown overnight in LB with double selection. Bacteriawere diluted to an OD600 of 0.1 and grown at 37C for 1 hour withchloramphenicol selection only. RFP expression was then induced using350 ng/mL of anhydrotetracycline and OD measurements were taken every 5minutes under continuous shaking in a BioTek Synergy 2 microplatereader.

C2c2 Nucleic Acid Preparation

The mammalian codon-optimized gene for C2c2 (Leptotrichia shahii) wassynthesized (GenScript) and cloned into a bacterial expression plasmid.E coli cells (BL21(DE3)) were transformed and grown overnight at 37° C.The protein was then purified using histidine-tags and Ni-NTA affinitycolumns and then further purified using FPLC gel filtration.

Nucleic acid templates for T7 transcription were synthesized from IDT.Templates for crRNAs were annealed to a short T7 primer and incubatedwith T7 polymerase overnight at 37° C. Templates for targeting innuclease assays were made double stranded using PCR and then incubatedwith T7 polymerase at 30° C. overnight.

5′ end labeling was accomplished using the 5′ oligonucleotide kit(VectorLabs) and with a maleimide-1R800 probe (Licor). 3′ end labelingwas performed using a 3′ oligonucleotide labeling kit (Sigma) usingddUTP-Cy5. Labeled probes were purified using Clean and Concentratorcolumns (Zymo).

C2c2 Protein Purification

The mammalian codon-optimized gene for C2c2 (Leptotrichia shahii) wassynthesized (GenScript) and cloned into a bacterial expression vector(6-His-MBP-TEV-Cpf1, a pET based vector kindly given to us by DougDaniels). 12 liters of Terrific Broth growth media with 100 μg/mLampicillin was inoculated with 10 mL overnight culture One Shot®BL21(DE3)pLysE (Invitrogen) cells containing the LshC2c2 expressionconstruct. Growth media plus inoculant was grown at 37° C. until thecell density reached 0.2 OD600, then the temperature was decreased to21° C. Growth was continued until OD600 reached 0.6 when a finalconcentration of 500 μM IPTG was added to induce MBP-C2c2 expression.The culture was induced for 14-18 hours before harvesting cells andfreezing at −80° C. until purification.

Cell paste was resuspended in 200 mL of Lysis Buffer (50 mM Hepes pH 7,2M NaCl, 5 mM MgCl2, 20 mM imidazole) supplemented with proteaseinhibitors (Roche cOmplete, EDTA-free) and lysozyme. Once homogenized,cells were lysed by sonication (Branson Sonifier 450) then centrifugedat 10,000 g for 1 hour to clear the lysate. The lysate was filteredthrough 0.22 micron filters (Millipore, Stericup) applied to a Ni-NTAsuperflow nickel resin (Qiagen), washed, and then eluted with a gradientof imidazole. Fractions containing protein of the expected size werepooled, TEV protease (Sigma) was added, and the sample was dialyzedovernight into TEV buffer (500 mM NaCl, 50 mM Hepes pH 7, 5 mM MgCl, 2mM DTT). After dialysis, TEV cleavage was confirmed by SDS-PAGE, and thesample was concentrated to 500 μL prior to loading on a gel filtrationcolumn (HiLoad 16/600 Superdex 200) via FPLC (AKTA Pure). Fractions fromgel filtration were analyzed by SDS-PAGE; fractions containing C2c2 werepooled and concentrated to 200 μL and either used directly forbiochemical assays or frozen at −80° C. for storage. Gel filtrationstandards were run on the same column equilibrated in 2M NaCl, Hepes pH7.0 to calculate the approximate size of LshC2c2.

Nucleic Acid Target Preparation

DNA oligo templates for T7 transcription were ordered from IDT.Templates for crRNAs were annealed to a short T7 primer and incubatedwith T7 polymerase overnight at 30° C. using the HiScribe T7 Quick HighYield RNA Synthesis kit (New England Biolabs). Target templates were PCRamplified to yield dsDNA and then incubated with T7 polymerase at 30° C.overnight using the same kit.

5′ end labeling was accomplished using the 5′ oligonucleotide kit(VectorLabs) and with a maleimide-IR800 probe (Licor). 3′ end labelingwas performed using a 3′ oligonucleotide labeling kit (Sigma) usingddUTP-Cy5. Labeled probes were purified using Clean and Concentratorcolumns (Zymo Research).

Nuclease Assay

Nuclease assays were performed with 160 nM of end-labeled ssRNA target,200 nM purified LshC2c2, and 100 nM crRNA, unless otherwise indicated,in nuclease assay buffer (40 mM Tris-HCl, 60 mM NaCl, 6 mM MgCl2, pH7.3). Reactions were allowed to proceed for 1 hour at 37° C. (unlessotherwise indicated) and were then quenched with proteinase K and EDTAfor 15 minutes at 37° C. The reactions were then denatured with 6M ureadenaturing buffer at 95° C. for 5 minutes. Samples were analyzed by gelelectrophoresis on 10% PAGE TBE-Urea run at 45° C. Gels were imagedusing a Licor Odyssey scanner.

Electrophoretic Mobility Shift Assay

Target ssRNA binding experiments were performed with a series ofhalf-log complex dilutions (crRNA and LshC2c2) from 2 μM to 0.2 μM (or 1μM to 0.1 μM in the case of R1278A LshC2c2). Binding assays wereperformed in nuclease assay buffer supplemented with 10 mM EDTA toprevent cutting, 5% glycerol, and 10 μg/mL heparin in order to avoidnon-specific interactions of the complex with target RNA. Reactions wereincubated at 37° C. for 20 minutes and then resolved on 6% PAGE TBE gelsat 4° C. (using 0.5×TBE buffer). Gels were imaged using the LicorOdyssey scanner.

NGS of In Vitro Cleaved RNA

In vitro nuclease assays were performed as described above usingunlabeled ssRNA targets. After one hour, samples were quenched withproteinase K+EDTA and then column purified (Zymo Clean andConcentrator). The RNA samples were then PNK and 5′ polyphosphatasetreated (Epicentre) before preparing a library for NGS using NEBNextSmall RNA Library Prep Set for Illumina sequencing. Libraries weresequenced on an Illumina MiSeq to sufficient depth and analyzed usingthe alignment tool BWA (H. Li, R. Durbin, Fast and accurate short readalignment with Burrows-Wheeler transform. Bioinformatics 25, 1754-1760(2009)).

In Vitro Co-Transcriptional DNA Cleavage Assay

The E. coli RNAP co-transcriptional DNA cleavage assay was performedessentially as described previously (Samai et al., Cell, 2015). Briefly,0.8 pmol of ssDNA template strand were annealed with 1.6 pmol of RNA intranscription buffer (from E. coli RNAP core enzyme New England Biolabs)without magnesium to prevent RNA hydrolysis. 0.75 ul of E. coli RNAPcore enzyme and Magnesium were added and the reaction incubated at 25°C. for 30 min and then transferred to 37° C. 1 pmol of freshly denaturednontemplate strand (NTS) were added and incubated at 37° C. for 15 minto obtain elongation complexes (ECs). 4 pmol of LshC2C2-crRNA complexesalong with 1.25 mM of RNTPs were added to the ECs and transcription wasallowed to proceed for 1 h at 37° C. DNA was resolved on a 10% PAGETBE-Urea gels following RNase and proteinase K treatment.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. A method of modifying a target locus of interest, the methodcomprising delivering to said locus a non-naturally occurring orengineered composition comprising a C2c2 effector protein and one ormore nucleic acid components, wherein the effector protein forms acomplex with the one or more nucleic acid components, the one or morenucleic acid components directs the complex to the target locus ofinterest and the complex binds to the target locus of interest.
 2. Themethod of claim 1, wherein the target locus of interest comprises RNA.3. The method of claim 1, wherein the modification of the target locusof interest comprises a nucleotide strand break.
 4. The method of claim1, wherein the C2c2 effector protein is codon optimized for expressionin a eukaryotic cell.
 5. The method of claim 1, wherein the C2c2effector protein is associated with one or more functional domains; andoptionally the effector protein contains one or more mutationsoptionally within an HEPN Domain, such as R597A, H602A, R1278A, and/orH1283A, whereby the complex can deliver an epigenentic modifier or atranscriptional or translational activation or repression signal.
 6. Themethod of claim 5, wherein the functional domain modifies transcriptionor translation of the target locus.
 7. The method of claim 1, whereinthe C2c2 effector protein comprises at least one or more nuclearlocalization signals.
 8. The method of claim 1, wherein the target locusof interest is provided via a nucleic acid molecule in vitro.
 9. Themethod of claim 1, wherein the target locus of interest is provided viaa nucleic acid molecule within a cell.
 10. The method of claim 9,wherein the cell comprises a prokaryotic cell.
 11. The method of claim9, wherein the cell comprises a eukaryotic cell.
 12. The method of claim1, wherein when in complex with the effector protein the nucleic acidcomponent(s) is capable of effecting sequence specific binding of thecomplex to a target sequence of the target locus of interest.
 13. Themethod of claim 1, wherein the nucleic acid component(s) comprise a dualdirect repeat sequence.
 14. The method of claim 1, wherein the effectorprotein and nucleic acid component(s) are provided via one or morepolynucleotide molecules encoding the polypeptides and/or the nucleicacid component(s), and wherein the one or more polynucleotide moleculesare operably configured to express the polypeptides and/or the nucleicacid component(s).
 15. The method of claim 14, wherein the one or morepolynucleotide molecules comprise one or more regulatory elementsoperably configured to express the polypeptides and/or the nucleic acidcomponent(s), optionally wherein the one or more regulatory elementscomprise a promoter(s) or inducible promotor(s).
 16. The method of claim14, wherein the one or more polynucleotide molecules are comprisedwithin one or more vectors.
 17. The method of claim 14, wherein the oneor more polynucleotide molecules are comprised within one vector. 18.The method of claim 16, wherein the one or more vectors comprise viralvectors.
 19. The method of claim 18, wherein the one or more viralvectors comprise one or more retroviral, lentiviral, adenoviral,adeno-associated or herpes simplex viral vectors.
 20. The method ofclaim 14, wherein the one or more polynucleotide molecules are comprisedin a delivery system.
 21. The method of claim 1, wherein thenon-naturally occurring or engineered composition is delivered via adelivery vehicle comprising liposome(s), particle(s), exosome(s),microvesicle(s), a gene-gun or one or more viral vector(s).
 22. Anon-naturally occurring or engineered composition which is a compositionhaving the characteristics as defined in claim
 1. 23. A non-naturallyoccurring or engineered composition comprising a C2c2 effector proteinand one or more nucleic acid components, wherein the effector proteinforms a complex with the one or more nucleic acid components, the one ormore nucleic acid components directs the complex to the target ofinterest and the complex binds to the target locus of interest.
 24. Thecomposition of claim 23, wherein the target locus of interest comprisesRNA.
 25. The composition of claim 23, wherein the modification of thetarget locus of interest comprises a nucleotide strand break.
 26. Thecomposition of claim 23, wherein the C2c2 effector protein is codonoptimized for expression in a eukaryotic cell.
 27. The composition ofclaim 23, wherein the C2c2 effector protein is associated with one ormore functional domains; and optionally the effector protein containsone or more mutations optionally within an HEPN Domain, such as R597A,H602A, R1278A, and/or H1283A, whereby the complex can deliver anepigenentic modifier or a transcriptional or translational activation orrepression signal.
 28. The composition of claim 27, wherein thefunctional domain modifies transcription or translation of the targetlocus.
 29. The composition of claim 23, wherein the C2c2 effectorprotein comprises at least one or more nuclear localization signals. 30.The composition of claim 23, wherein the target locus of interest iscomprised in a nucleic acid molecule in vitro.
 31. The composition ofclaim 23, wherein the target locus of interest is comprised in a nucleicacid molecule within a cell.
 32. The composition of claim 31, whereinthe cell comprises a prokaryotic cell or a eukaryotic cell. 33.(canceled)
 34. The composition of claim 23, wherein when in complex withthe effector protein the nucleic acid component(s) is capable ofeffecting sequence specific binding of the complex to a target sequenceof the target locus of interest.
 35. The composition of claim 23,wherein the nucleic acid component(s) comprise a dual direct repeatsequence.
 36. The composition of claim 23, wherein the effector proteinand nucleic acid component(s) are provided via one or morepolynucleotide molecules encoding the polypeptides and/or the nucleicacid component(s), and wherein the one or more polynucleotide moleculesare operably configured to express the polypeptides and/or the nucleicacid component(s).
 37. The composition of claim 36, wherein the one ormore polynucleotide molecules comprise one or more regulatory elementsoperably configured to express the polypeptides and/or the nucleic acidcomponent(s), optionally wherein the one or more regulatory elementscomprise a promoter(s) or inducible promotor(s).
 38. The composition ofclaim 36, wherein the one or more polynucleotide molecules are comprisedwithin one or more vectors.
 39. The composition of claim 36, wherein theone or more polynucleotide molecules are comprised within one vector.40. The composition of claim 38, wherein the one or more vectorscomprise viral vectors.
 41. The composition of claim 40, wherein the oneor more viral vectors comprise one or more retroviral, lentiviral,adenoviral, adeno-associated or herpes simplex viral vectors.
 42. Thecomposition of claim 36, wherein the one or more polynucleotidemolecules are comprised in a delivery system.
 43. The composition ofclaim 23, wherein the non-naturally occurring or engineered compositionis delivered via a delivery vehicle comprising liposome(s), particle(s),exosome(s), microvesicle(s), a gene-gun or one or more viral vector(s).44. A vector system comprising one or more vectors, the one or morevectors comprising one or more polynucleotide molecules encodingcomponents of a non-naturally occurring or engineered composition whichis a composition having the characteristics as defined in claim
 23. 45.A delivery system configured to deliver a C2c2 effector protein and oneor more nucleic acid components of a non-naturally occurring orengineered composition which is a composition having the characteristicsas defined in claim
 23. 46. The delivery system of claim 45, whichcomprises one or more vectors or one or more polynucleotide molecules,the one or more vectors or polynucleotide molecules comprising one ormore polynucleotide molecules encoding the C2c2 effector protein and oneor more nucleic acid components of the non-naturally occurring orengineered composition having the characteristics as defined in any oneof the preceding claims.
 47. The non-naturally occurring or engineeredcomposition, vector system, or delivery system of claim 23, for use in atherapeutic method of treatment.
 48. A cell modified according to themethod, or engineered to comprise or express, optionally inducibly orconstituently, the composition or a component thereof of claim
 23. 49.The cell according to claim 48, wherein the modification results in: thecell comprising altered transcription or translation of at least one RNAproduct; the cell comprising altered transcription or translation of atleast one RNA product, wherein the expression of the at least oneproduct is increased; or the cell comprising altered transcription ortranslation of at least one RNA product, wherein the expression of theat least one product is decreased.
 50. The cell of claim 49, wherein thecell comprises a eukaryotic cell or a prokaryotic cell.
 51. The cellaccording to claim 48, wherein the cell comprises a mammalian cell. 52.(canceled)
 53. The non-naturally occurring or engineered composition,vector system, or delivery system of claim 23, for use in: RNA sequencespecific interference, RNA sequence specific gene regulation, screeningof RNA or RNA products or lincRNA or non-coding RNA, or nuclear RNA, ormRNA, mutagenesis, Fluorescence in situ hybridization, breeding, invitro or in vivo induction of cell dormancy, in vitro or in vivoinduction of cell cycle arrest, in vitro or in vivo reduction of cellgrowth and/or cell proliferation, in vitro or in vivo induction of cellanergy, in vitro or in vivo induction of cell apoptosis, in vitro or invivo induction of cell necrosis, in vitro or in vivo induction of celldeath, or in vitro or in vivo induction of programmed cell death.
 54. Acell line of or comprising the cell according to claim 48, or progenythereof.
 55. A multicellular organism comprising one or more cellsaccording to claim
 50. 56. A plant or animal model comprising one ormore cells according to claim 48; said cell(s) optionally inducibly orconstituently expressing the composition or a component thereof of anyone of the preceding claims.
 57. A product from a cell of claim
 23. 58.The product of claim 57, wherein the amount of product is greater thanor less than the amount of product from a cell that has not hadalteration or modification by a method or composition of any claim 23.59. The product of claim of claim 57, wherein the product is altered incomparison with the product from a cell that has not had alteration ormodification by a method or composition of claim
 23. 60. An assay,screening method or mutagenesis method comprising a system or method orcells of claim
 23. 61. In an RNA-based assay, screening method ormutagenesis method wherein the improvement comprises, instead of usingRNA, the method comprises using a composition as in claim
 23. 62. Themethod of claim 61 wherein the RNA-based assay, screening method ormutagenesis method is an RNAi or Fluorescence in situ hybridizationmethod.
 63. Use of the non-naturally occurring or engineeredcomposition, vector system, or delivery system of claim 23, for: RNAsequence specific interference, RNA sequence specific gene regulation,screening of RNA or RNA products or lincRNA or non-coding RNA, ornuclear RNA, or mRNA, mutagenesis, Fluorescence in situ hybridization,breeding, in vitro or in vivo induction of cell dormancy, in vitro or invivo induction of cell cycle arrest, in vitro or in vivo reduction ofcell growth and/or cell proliferation, in vitro or in vivo induction ofcell anergy, in vitro or in vivo induction of cell apoptosis, in vitroor in vivo induction of cell necrosis, in vitro or in vivo induction ofcell death, or in vitro or in vivo induction of programmed cell death.64. The method according to claim 1, wherein said method results in: RNAsequence specific interference, RNA sequence specific gene regulation,screening of RNA or RNA products or lincRNA or non-coding RNA, ornuclear RNA, or mRNA, mutagenesis, Fluorescence in situ hybridization,breeding, in vitro or in vivo induction of cell dormancy, in vitro or invivo induction of cell cycle arrest, in vitro or in vivo reduction ofcell growth and/or cell proliferation, in vitro or in vivo induction ofcell anergy, in vitro or in vivo induction of cell apoptosis, in vitroor in vivo induction of cell necrosis, in vitro or in vivo induction ofcell death, or in vitro or in vivo induction of programmed cell death.65. A method for: RNA sequence specific interference, RNA sequencespecific gene regulation, screening of RNA or RNA products or lincRNA ornon-coding RNA, or nuclear RNA, or mRNA, mutagenesis, Fluorescence insitu hybridization, breeding, in vitro or in vivo induction of celldormancy, in vitro or in vivo induction of cell cycle arrest, in vitroor in vivo reduction of cell growth and/or cell proliferation, in vitroor in vivo induction of cell anergy, in vitro or in vivo induction ofcell apoptosis, in vitro or in vivo induction of cell necrosis, in vitroor in vivo induction of cell death, or in vitro or in vivo induction ofprogrammed cell death comprising introducing or inducing in vitro or invivo in a target cell the non-naturally occurring or engineeredcomposition, vector system, or delivery system of claim
 23. 66. Anengineered, non-naturally occurring CRISPR-Cas system comprising one ormore vectors comprising: a) a first regulatory element operable in aeukaryotic or prokaryotic cell operably linked to at least onenucleotide sequence encoding a CRISPR-Cas system guide RNA thathybridizes with a target sequence of an RNA molecule encoded by a DNAmolecule in a eukaryotic or prokaryotic cell, wherein the DNA moleculeencodes and the eukaryotic or prokaryotic cell expresses at least onegene product, and b) a second regulatory element operable in aeukaryotic or prokaryotic cell operably linked to a nucleotide sequenceencoding a Type-II C2c2 effector protein, wherein components (a) and (b)are located on same or different vectors of the system, whereby theguide RNA targets and hybridizes with the target sequence and the C2c2effector protein cleaves the RNA molecule, whereby expression of the atleast one gene product is altered; and, wherein the C2c2 effectorprotein and the guide RNA do not naturally occur together.
 67. Anengineered, non-naturally occurring composition comprising a CRISPR-Cassystem, said system comprising a functional CRISPR C2c2 effector proteinand guide RNA (gRNA); wherein the gRNA comprises a dead guide sequence;whereby the gRNA is capable of hybridizing to a target sequence; wherebythe CRISPR-Cas system is directed to the target sequence with reducedindel activity resultant from nuclease activity of a non-mutant C2c2effector protein of the system.
 68. A method of inhibiting cell growth,the method comprising delivering to the cell a non-naturally occuring orengineered composition comprising a functional CRISPR C2c2 effectorprotein and guide RNA (gRNA); whereby the gRNA is capable of hybridizingto a target RNA sequence of the cell; whereby the CRISPR-Cas system isdirected to the target RNA sequence with reduced indel activityresultant from nuclease activity of a non-mutant C2c2 effector proteinof the system.
 69. A CRISPR associated Cas vector system comprising oneor more vectors comprising: a) a first regulatory element operable in aeukaryotic or prokaryotic cell operably linked to at least onenucleotide sequence encoding a CRISPR-Cas system guide RNA thathybridizes with a target sequence of an RNA molecule encoded by a DNAmolecule in a eukaryotic or prokaryotic cell, wherein the DNA moleculeencodes and the eukaryotic or prokaryotic cell expresses at least onegene product, and b) a second regulatory element operable in aeukaryotic or prokaryotic cell operably linked to a nucleotide sequenceencoding a Type-II C2c2 effector protein, wherein components (a) and (b)are located on same or different vectors of the system, whereby theguide RNA targets and hybridizes with the target sequence and the C2c2effector protein cleaves the RNA molecule, whereby expression of the atleast one gene product is altered; and, wherein the C2c2 effectorprotein and the guide RNA do not naturally occur together.